Figure 2A - E . The PFC was stained with CB1 receptor ( A1 - D1 , green ) or 5HT6 receptor ( E1 - H1 , green ) , Bassoon ( A2 and E2 , red ) , GAD65 ( B2 and F2 , red ) , PSD - 95 ( C2 and G2 , red ) , or SERT ( D2 and H2 , red ) antibodies . Merge images are also depicted ( A3 - H3 ) . CB1 receptors were mainly co - localized with the presynaptic marker Bassoon ( white arrows , A3 ) , mostly within GABAergic boutons ( white arrows , B3 ) and to a lesser extend with PSD - 95 ( white arrows , C3 ) and SERT ( D3 ) , while 5 - HT6 receptors were mainly co - localized with PSD - 95 ( white arrows , G3 ) . No co - localization of CB1 and 5 - HT6 receptors was detected ( white arrows indicate the 5 - HT6 staining , H3 ) . Scale bar : 10 μm . The scatter plot represents the co - localization analysis for CB1 and 5 - HT6 receptor immunostainings . Graph on the top , Mander ' s split coefficient was used to identify the fraction of CB1 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 66 ± 0 . 02 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 56 ± 0 . 01 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 15 ± 0 . 01 , n = 4 ) or SERT ( Mander ' s coefficient : 0 . 082 ± 0 . 004 , n = 4 ) . Graph on the bottom . Mander ' s split coefficient was used to identify the fraction of 5 - HT6 receptors that co - localizes with Bassoon ( Mander ' s coefficient : 0 . 09 ± 0 . 01 , n = 4 ) , GAD65 ( Mander ' s coefficient : 0 . 059 ± 0 . 004 , n = 4 ) , PSD95 ( Mander ' s coefficient : 0 . 58 ± 0 . 01 , n = 4 ) or CB1 receptors ( Mander ' s coefficient : 0 . 0020 ± 0 . 0004 , n = 4 ) . Error bars correspond to the mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-E. The PFC was stained with CB1 receptor (A1-D1, green) or 5HT6 receptor (E1-H1, green), Bassoon (A2 and E2, red), GAD65 (B2 and F2, red), PSD-95 (C2 and G2, red), or SERT (D2 and H2, red) antibodies. Merge images are also depicted (A3-H3). CB1 receptors were mainly co-localized with the presynaptic marker Bassoon (white arrows, A3), mostly within GABAergic boutons (white arrows, B3) and to a lesser extend with PSD-95 (white arrows, C3) and SERT (D3), while 5-HT6 receptors were mainly co-localized with PSD-95 (white arrows, G3). No co-localization of CB1 and 5-HT6 receptors was detected (white arrows indicate the 5-HT6 staining, H3). Scale bar: 10 μm. The scatter plot represents the co-localization analysis for CB1 and 5-HT6 receptor immunostainings. Graph on the top, Mander's split coefficient was used to identify the fraction of CB1 receptors that co-localizes with Bassoon (Mander's coefficient: 0.66 ± 0.02, n=4), GAD65 (Mander's coefficient: 0.56 ± 0.01, n=4), PSD95 (Mander's coefficient: 0.15 ± 0.01, n=4) or SERT (Mander's coefficient: 0.082 ± 0.004, n=4). Graph on the bottom. Mander's split coefficient was used to identify the fraction of 5-HT6 receptors that co-localizes with Bassoon (Mander's coefficient: 0.09 ± 0.01, n=4), GAD65 (Mander's coefficient: 0.059 ± 0.004, n=4), PSD95 (Mander's coefficient: 0.58 ± 0.01, n=4) or CB1 receptors (Mander's coefficient: 0.0020 ± 0.0004, n=4). Error bars correspond to the mean ± SEM.


Figure 3B . Top : representative Western blots assessing mTOR activity in PFC are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected mice . They are the means ± S . E . M . of results obtained in six mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test . C . The plots represent the discrimination index measured in each condition . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test . The discrimination index for the novel object recognition task is 0 . 34 ± 0 . 06 ( N = 14 ) and - 0 . 08 ± 0 . 07 ( N = 20 ) , for mice injected with Vehicle and THC , respectively , p < 0 . 001 and - 0 . 09 ± 0 . 08 ( N = 21 ) and 0 . 00 ± 0 . 06 ( N = 18 ) , for mice treated with THC + SB258285 and THC + Rapa , respectively , p > 0 . 05 vs . THC - injected mice ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index equal to zero ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Top: representative Western blots assessing mTOR activity in PFC are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected mice. They are the means ± S.E.M. of results obtained in six mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.C. The plots represent the discrimination index measured in each condition. *** p < 0.001, one-way ANOVA followed by Bonferroni test. The discrimination index for the novel object recognition task is 0.34 ± 0.06 (N=14) and -0.08 ± 0.07 (N= 20), for mice injected with Vehicle and THC, respectively, p < 0.001 and -0.09 ± 0.08 (N=21) and 0.00 ± 0.06 (N=18), for mice treated with THC+SB258285 and THC+Rapa, respectively, p > 0.05 vs. THC-injected mice (error bars correspond to the mean ± SEM, the dotted line to a discrimination index equal to zero).


Figure 4B . Left : representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of GABA mIPSC frequency and amplitude measured during the last minute of recording . The mIPSC frequency is : 3 . 2 ± 0 . 4 Hz and 1 . 3 ± 0 . 2 Hz for Vehicle ( n = 9 from N = 4 ) and THC ( n = 7 from N = 4 ) conditions , respectively , p < 0 . 001 and 2 . 5 ± 0 . 2 Hz and 4 . 1 ± 0 . 4 Hz for THC + SB258585 ( n = 8 from N = 4 ) and THC + Rapa ( n = 8 from N = 4 ) conditions , respectively , p < 0 . 01 and p < 0 . 001 vs . THC - injected mice . C . Left : representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated . Right : the histograms represent means ± S . E . M . of AMPA mEPSC frequency and amplitude measured during the last minute of recording , n = neurons from 3 to 5 mice per group . The mEPSC frequency is : 4 . 7 ± 0 . 4 Hz for Vehicle ( n = 9 from N = 7 ) and 6 . 8 ± 0 . 4 Hz for THC ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 01and 5 . 3 ± 0 . 5 Hz and 4 . 6 ± 0 , 3 Hz for THC + SB258585 ( n = 10 from N = 6 ) and THC + Rapa ( n = 9 from N = 5 ) conditions , respectively , p < 0 . 05 and p < 0 . 01 vs . THC - injected mice . In B and C , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , one - way ANOVA followed by Newman - Keuls test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Left: representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of GABA mIPSC frequency and amplitude measured during the last minute of recording. The mIPSC frequency is: 3.2 ± 0.4 Hz and 1.3 ± 0.2 Hz for Vehicle (n=9 from N= 4) and THC (n=7 from N= 4) conditions, respectively, p < 0.001 and 2.5 ± 0.2 Hz and 4.1 ± 0.4 Hz for THC+SB258585 (n=8 from N= 4) and THC+Rapa (n=8 from N= 4) conditions, respectively, p < 0.01 and p < 0.001 vs. THC-injected mice.C. Left: representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of AMPA mEPSC frequency and amplitude measured during the last minute of recording, n = neurons from 3 to 5 mice per group. The mEPSC frequency is: 4.7 ± 0.4 Hz for Vehicle (n=9 from N= 7) and 6.8 ± 0.4 Hz for THC (n=9 from N= 5) conditions, respectively, p < 0.01and 5.3 ± 0.5 Hz and 4.6 ± 0,3 Hz for THC+SB258585 (n=10 from N= 6) and THC+Rapa (n=9 from N= 5) conditions, respectively, p < 0.05 and p < 0.01 vs. THC-injected mice. In B and C, * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Newman-Keuls test.


Figure 5A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . A . The resting membrane potential ( RMP ) was determined immediately after whole - cell formation : - 71 . 8 ± 1 . 2 mV and - 60 . 1 ± 1 . 5 mV for Vehicle and THC conditions , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . B . Action potentials were evoked by a ramp current injection with a 10 pA - step for 2 ms and only the first action potential ( AP ) were used to estimate the AP threshold : - 34 . 1 ± 0 . 7 mV and - 39 . 7 ± 0 . 8 mV for Vehicle - and THC - injected mice , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . C . The rheobase represents the minimal current required to induce neuronal firing , it was lowered in THC condition : 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions , respectively , p < 0 . 001 . A - D . Mice were injected daily with THC ( 5 mg / kg ) or vehicle ( Veh ) during adolescence , from PND 30 to 45 . SB258585 ( SB , 2 . 5 mg / kg ) or rapamycin ( Rapa , 1 . 5 mg / kg ) were administered concomitantly with THC ( n = 20 from N = 5 ) or vehicle ( n = 18 from N = 6 ) . Electrophysiological recordings were performed from PND 60 . D . The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs . E . top : Voltage sag in response to hyperpolarizing current injection ( 50 pA - increments , from - 400 to 0 pA ) in PFC pyramidal neurons from mice injected with vehicle ( Veh , n = 10 from N = 5 ) , THC ( n = 11 from N = 5 ) , THC + SB258585 ( THC / SB , n = 7 from N = 4 ) or THC + Rapa ( THC / Rapa , n = 7 from N = 4 ) during adolescence . * * * p < 0 . 001 , one - way ANOVA followed by Bonferroni test , vs . THC - injected mice . Errors bars correspond to the mean ± SEM . Voltage sag is indicated by arrowheads . F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( F ) resting potential membrane : THC + ZD : - 74 . 14 ± 1 . 70 mV , p < 0 . 001 vs . THC condition , THC + DDOA : - 70 . 47 ± 1 . 19 mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 63 . 10 ± 0 . 81 mV , p < 0 . 001 vs . THC condition , F - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( G ) AP threshold : THC + ZD : - 33 . 59 ± 2 . 96 mV , p < 0 . 01 vs . THC condition , THC + DDOA : - 34 . 28 ± 0 . 98mV , p < 0 . 001 vs . THC condition , THC + ODQ : - 36 . 84 ± 0 . 79 mV , p < 0 . 001 vs . THC conditionF - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( H ) rheobase : THC + ZD : 806 ± 63 pA , p < 0 . 001 vs . THC condition , THC + DDOA : 607 ± 43 pA , p < 0 . 001 vs . THC condition , THC + ODQ : 422 ± 34 pA , p < 0 . 001 vs . THC conditionF - I . Effect of ZD7288 ( ZD , 10 μM , n = 10 from N = 3 ) , DDOA ( 15 μM , n = 9 from N = 4 ) or ODQ ( 10 μM , n = 12 from N = 4 ) on intrinsic properties of PFC pyramidal neurons . THC and vehicle conditions are similar to those on panel A - D . ( I ) firing rate . Note that Vehicle - and THC - injected mice are the same as those used in experiments illustrated on Figure 5A - D . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , n . s . p > 0 . 05 one - way ANOVA followed by Bonferroni test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. A. The resting membrane potential (RMP) was determined immediately after whole-cell formation: -71.8 ± 1.2 mV and -60.1 ± 1.5 mV for Vehicle and THC conditions, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. B. Action potentials were evoked by a ramp current injection with a 10 pA-step for 2 ms and only the first action potential (AP) were used to estimate the AP threshold: -34.1 ± 0.7 mV and -39.7 ± 0.8 mV for Vehicle- and THC-injected mice, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. C. The rheobase represents the minimal current required to induce neuronal firing, it was lowered in THC condition: 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions, respectively, p < 0.001.A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. D. The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs.E. top: Voltage sag in response to hyperpolarizing current injection (50 pA-increments, from -400 to 0 pA) in PFC pyramidal neurons from mice injected with vehicle (Veh, n= 10 from N=5), THC (n = 11 from N=5), THC+SB258585 (THC/SB, n=7 from N=4) or THC+Rapa (THC/Rapa, n=7 from N=4) during adolescence. *** p < 0.001, one-way ANOVA followed by Bonferroni test, vs. THC-injected mice. Errors bars correspond to the mean ± SEM. Voltage sag is indicated by arrowheads.F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (F) resting potential membrane: THC + ZD: -74.14 ± 1.70 mV, p < 0.001 vs. THC condition, THC + DDOA: -70.47 ± 1.19 mV, p < 0.001 vs. THC condition, THC + ODQ: -63.10 ± 0.81 mV, p < 0.001 vs. THC condition,F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (G) AP threshold: THC + ZD: -33.59 ± 2.96 mV, p < 0.01 vs. THC condition, THC + DDOA: -34.28 ± 0.98mV, p < 0.001 vs. THC condition, THC + ODQ: -36.84 ± 0.79 mV, p < 0.001 vs. THC conditionF-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (H) rheobase: THC + ZD: 806 ± 63 pA, p < 0.001 vs. THC condition, THC + DDOA: 607 ± 43 pA, p < 0.001 vs. THC condition, THC + ODQ: 422 ± 34 pA, p < 0.001 vs. THC conditionF-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (I) firing rate. Note that Vehicle- and THC-injected mice are the same as those used in experiments illustrated on Figure 5A-D. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s. p > 0.05 one-way ANOVA followed by Bonferroni test.


Figure 6A . In left , normalized peak amplitudes of isolated AMPA EPSCs recorded at - 60 mV , before and after pairing protocol , are illustrated . Representative traces of AMPA EPSCs before ( 1 ) or after ( 2 ) the pairing protocol are also illustrated ( right panel ) for each experimental condition : Vehicle - injected mice ( white circles , n = 3 from N = 3 ) , THC - injected mice ( green circles , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black circles , n = 3 from N = 3 ) . Errors bars correspond to mean ± SEM . B . The histogram represents the means ± SEM of AMPA EPSCs in % of baseline , measured during the last 5 min of baseline or the last 5 min of recording , for each experimental condition : vehicle - injected mice ( white bar , n = 3 from N = 3 ) , THC - injected mice ( green bar , n = 3 from N = 3 ) and THC - injected mice treated with SB258585 during adolescence ( black bar , n = 3 from N = 3 ) , n . s . p > 0 . 05 , * p < 0 . 05 , one - way ANOVA followed by Tukey test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. In left, normalized peak amplitudes of isolated AMPA EPSCs recorded at -60 mV, before and after pairing protocol, are illustrated. Representative traces of AMPA EPSCs before (1) or after (2) the pairing protocol are also illustrated (right panel) for each experimental condition: Vehicle-injected mice (white circles, n=3 from N=3), THC-injected mice (green circles, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black circles, n=3 from N=3). Errors bars correspond to mean ± SEM.B. The histogram represents the means ± SEM of AMPA EPSCs in % of baseline, measured during the last 5 min of baseline or the last 5 min of recording, for each experimental condition: vehicle-injected mice (white bar, n=3 from N=3), THC-injected mice (green bar, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black bar, n=3 from N=3), n.s. p > 0.05, * p< 0.05, one-way ANOVA followed by Tukey test.


Figure 1 ( A ) Representative kymographs showing tetramethylrhodamine ( TMR ) - labeled DDB movement on each type of recombinant MT lattice , as indicated . Scale bars , 5 μm and 25 sec . Pink arrowhead marks a diffusive DDB complex . See also Movie EV1 . ( B ) Quantification of DDB velocity on MT lattices lacking each of the tubulin - CTTs ( n ≥ 100 DDB complexes per condition from at least three independent experiments ) . No statistical difference was observed between WT and mutant MTs ( P ≥ 0 . 1 , unpaired t test ) ( C - D ) Quantification of the number of processive ( C ) , or diffusive ( D ) DDB complexes relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Processive ( unidirectional motion exceeding 0 . 5 μm ) and diffusive ( back - and - forth motion ) events were scored in a field of view and then divided by the lengths of MTs and time , thus normalizing to a value of number of motility events per μm MT per sec . Error bars SD . ( E - F ) TIRF images of 1 nM TMR - labeled GST - hDyn ( 2 mM ATP ) ( E ) , or 0 . 3 nM TMR - labeled p150 ( F ) molecules ( green ) bound to either Δα - CTT ( top ) , or Δβ - CTT MTs ( bottom ) . Pink arrows show mutant MTs in both the raw , and SD map images , highlighting differences in binding between WT and mutant MTs that are apparent in the SD map . Right , standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities ( see Methods ) . Scale bars are 5 μm . ( G ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . Image shows first frame from time series . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD . P value ( unpaired t test ) of Δα - CTT vs . Δβ - CTT MTs = 0 . 5208 for GST - hDyn , and ≤ 0 . 0001 for p150 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD.(E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.


Figure 2 ( A ) TIRF image of TMR - labeled DDB molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . A standard deviation map of the whole time sequence is shown , revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs ( pink arrow ) . Representative kymographs of DDB movement are shown from each type of MT present in the same chamber . Scale bars are 5 μm and 25 sec . See also Movie EV2 . ( B - C ) Quantification of the number of processive ( B ) or diffusive ( C ) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber ( n ≥ 20 MTs quantified for each condition from three independent experiments ) . Error bars represent SD . Data from Δα - CTT MTs ( Fig . 1 ) are re - plotted for comparison . ( D ) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs . Data points from two independent experiments are shown . ( E ) TIRF images of 0 . 3 nM TMR - labeled p150 ( top ) or 1 nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either tyrosinated ( red ) or detyrosinated ( blue ) MTs . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown . Scale bars are 5 μm and 25 sec . ( F ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2 mM ATP ) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT . ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown.(E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


Figure 3 ( A ) Immunoblot of brain tubulin with or without carboxypeptidase A ( CPA ) . Note CPA specifically removes the α - tubulin C - terminal tyrosine without affecting other PTMs . ( B ) TIRF image of TMR - labeled DDB molecules ( green ) on either WT ( red ) or CPA - treated ( blue ) pig brain MTs . Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec . ( C ) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA - treated MTs , relative to WT MTs in the same chamber ( n ≥ 7 MTs of each type quantified from at least three independent experiments ) . Error bars represent SD . ( D ) TIRF images of 0 . 3nM TMR - labeled p150 ( top ) or 1nM TMR - labeled GST - hDyn ( bottom ) molecules ( green ) bound to either WT ( red ) , or CPA - treated MTs ( blue ) . Right , standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA - treated are shown . Scale bars are 5 μm and 25 sec for p150 , and 5 μm and 12 sec for GST - hDyn ( E ) Quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for TMR - labeled GST - hDyn ( 2mM ATP ) or p150 bound to CPA - treated MTs relative to WT MTs in the same chamber . The intensity of GST - hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST - hDyn to the MT ( n ≥ 10 MTs quantified for each condition from at least three independent experiments ) . Error bars represent SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunoblot of brain tubulin with or without carboxypeptidase A (CPA). Note CPA specifically removes the α-tubulin C-terminal tyrosine without affecting other PTMs.(B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD.(D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.


Figure 4 ( A ) Schematic of the p150 constructs used . The p135 isoform lacks the CAP - Gly domain , but retains part of the adjacent basic domain ( red ) and coiled - coil domains ( blue ) . Right , Coomassie blue stained gel of the purified p150 and p135 proteins . Molecular weight markers are indicated . ( B ) TIRF image of either p150 ( 0 . 3 nM ) , or p135 ( 0 . 3 or 6 nM ) bound to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Note : no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform . Proteins were visualized by fluorescence from the GFP tag fused to each construct . ( C ) Still images from a TIRF time - series of p135 ( green ) binding to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Right , standard deviation map of p135 binding shows no preference for either type of MT , and quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for p135 bound to CPA - treated MTs relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from three independent experiments ) . Scale bars are 5 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Schematic of the p150 constructs used. The p135 isoform lacks the CAP-Gly domain, but retains part of the adjacent basic domain (red) and coiled-coil domains (blue). Right, Coomassie blue stained gel of the purified p150 and p135 proteins. Molecular weight markers are indicated.(B) TIRF image of either p150 (0.3 nM), or p135 (0.3 or 6 nM) bound to either WT (red), or CPA-treated porcine MTs (blue). Note: no binding of p135 is observed at comparable concentrations to p150 indicating the lower binding affinity of this isoform. Proteins were visualized by fluorescence from the GFP tag fused to each construct.(C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.


Figure 5 ( B ) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated ( red ) and detyrosinated ( blue ) sections . Most TMR - labeled DDB molecules ( green ) move processively through the annealed junction between the two types of MT lattice ( 65 % , n = 836 processive complexes , 65 MT - MT junctions , three independent experiments ) . Diffusive DDB molecules ( white arrow ) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary . Right , video frames showing a processive DDB complex ( pink arrowhead ) moving across the junction ( yellow arrowhead ) of tyrosinated and detryosinated MT . The ( + ) and ( - ) signs denote MT polarity inferred from the directionality of DDB movement . Scale bars are 5 μm and 25 sec . See also Movie EV3 . ( C ) TIRF images of low ( 0 . 3 nM ) and high ( 10 nM ) concentrations of TMR - labeled p150 ( green ) bound to chimeric yeast MTs containing tyrosinated ( red ) and detyrosinated ( blue ) sections of MT . Representative kymographs below show diffusive behavior of p150 molecules on the MT . Note that p150 rarely diffuses into the detyrosinated section of MT , even at high concentrations . Scale bars are 5 μm and 10 sec . ( D - E ) TIRFimages and kymographs of annealed WT and CPA - treated porcineMTs ( D ) , or annealed WT MTs ( E ) . The majority of DDB complexes ( 78 % , n = 960 processive complexes , 45 MT - MT junctions , three independent experiments ) move processively across the boundary between WT and CPA - MTs ( D ) . Note TMR - labeled DDB complexes ( green ) in ( E ) traverse the annealed boundary without pausing ( 93 % , n = 677 processive complexes , 45 MT - MT junctions , three independent experiments ) , suggesting that the annealed junction does not affect DDB motility . ( F ) TIRF image and kymograph from a chimeric MT composed of WT ( red ) and subtilisin - treated porcine MTs ( blue ) . Note some DDB complexes ( pink arrows ) traverse the boundary and continue moving along the subtilisin - treated section of the MT ( 41 % , n = 709 processive complexes , 56 MT - MT junctions , 3 independent experiments ) . All scale bars in D - F are 5 μm and 25 sec .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.(F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.


Figure 1A . Heatmap of IC50 values for cell viability . Human dermal fibroblasts ( HDFB ) controls and 4 MCC cell lines ( PeTa , MKL - 1 , WaGa , MS - 1 ) were treated with the 43 indicated small molecules targeting epigenetic modifiers . IC50 values are depicted as log10 ( IC50 ) in [ mM ] . n / a , IC50 values could not be calculated . n = 4 technical replicates . B . Dose - response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK - LSD1 or ORY - 1001 . Dose - response curves of 3 other MCC cell lines are displayed in Fig EV1A . n = 4 technical replicates . Data are represented as means ± SD . D . In vitro competition assay of the 3 MCC cell lines MKL - 2 , PeTa , and WaGa transduced with either shLSD1 . 1 , shLSD1 . 2 , shRenilla ( negative control ) , or shRPS15 ( positive control ) . Individual graphs are displayed in Fig EV1E . E . Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa , MKL - 1 , MKL - 2 of the genes targeted by the compound library in Fig 1A . A score of 0 indicates that a gene is not essential ; correspondingly - 1 is comparable to the median of all pan - essential genes . Data obtained from DepMap ; dependencies for the individual cell lines are displayed in Fig EV1F . F . Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues , ordered according to mean dependency score . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Heatmap of IC50 values for cell viability. Human dermal fibroblasts (HDFB) controls and 4 MCC cell lines (PeTa, MKL-1, WaGa, MS-1) were treated with the 43 indicated small molecules targeting epigenetic modifiers. IC50 values are depicted as log10(IC50) in [mM]. n/a, IC50 values could not be calculated. n = 4 technical replicates.B. Dose-response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK-LSD1 or ORY-1001. Dose-response curves of 3 other MCC cell lines are displayed in Fig EV1A. n = 4 technical replicates. Data are represented as means ± SD.D. In vitro competition assay of the 3 MCC cell lines MKL-2, PeTa, and WaGa transduced with either shLSD1.1, shLSD1.2, shRenilla (negative control), or shRPS15 (positive control). Individual graphs are displayed in Fig EV1E.E. Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa, MKL-1, MKL-2 of the genes targeted by the compound library in Fig 1A. A score of 0 indicates that a gene is not essential; correspondingly -1 is comparable to the median of all pan-essential genes. Data obtained from DepMap; dependencies for the individual cell lines are displayed in Fig EV1F.F. Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues, ordered according to mean dependency score. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.


Figure 2 B . Individual tumor growth in GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 22 C . Representative picture of mice treated with vehicle or GSK - LSD1 at day 41 after PeTa cell injection . Tumor location is indicated with a circle D . Kaplan - Meier curve of GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or the greatest dimension was ≥ 1 . 5cm . Red arrow : start of therapy , day 22 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test ) F . Individual tumor growth in GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 1 G . Relative mouse weight ( % ) during treatment of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice H . Kaplan - Meier curve of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or greatest dimension ≥ 1 . 5cm . Red arrow : start of therapy , day 1 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 B. Individual tumor growth in GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 22  C. Representative picture of mice treated with vehicle or GSK-LSD1 at day 41 after PeTa cell injection. Tumor location is indicated with a circle  D. Kaplan-Meier curve of GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or the greatest dimension was ≥ 1.5cm. Red arrow: start of therapy, day 22. ****p < 0.0001 (log-rank Mantel-Cox test)  F. Individual tumor growth in GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 1  G. Relative mouse weight (%) during treatment of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice  H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test)


Figure 3A . Ki - 67 staining of PeTa cells treated for 3 days with GSK - LSD1 ( 1 μM ) or ORY - 1001 ( 1 μM ) . n = 3 biological replicates . Data are represented as means ± SD . * * * p < 0 . 001 ( DMSO vs GSK - LSD1 p = 0 . 0002 ; DMSO vs ORY - 1001 p = 0 . 0004 ; unpaired Student ' s t - test ) . B . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK - LSD1 or DMSO treatment . C . Quantification of EdU / Propidium iodide staining depicted in Fig 3B . n = 3 biological replicates . Data are represented as means ± SD . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; ns , non - significant ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0059 ; S phase : DMSO vs GSL - LSD1 p < 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 2338 ; unpaired Student ' s t - test ) . D . Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment . Etoposide serves as positive control for apoptosis , H3 serves as loading control . c . PARP1 , cleaved PARP1 . E . Caspase - 3 / 7 cleavage activity for PeTa cell line after 6 days of indicated treatment . Etoposide serves as a positive control for apoptosis . n = 3 technical replicates . Data are represented as means ± SD . RLU , relative luminescence units . F . Representative FACS readout of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment after 6 days . G . Quantification of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment at indicated time points . H . Representative FACS readout of Annexin V / DAPI staining of PeTa cells after 6 days of 100 nM GSK - LSD1 DMSO treatment . I . Quantification of Annexin V / DAPI staining depicted in Fig 3H . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( Early , Late , Total ; unpaired Student ' s t - test ) . J . Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK - LSD1 or vehicle treatment . Representative images in Appendix Fig S1B . n = 8 , * * * p = 0 . 001 ( Vehicle vs GSK - LSD1 ; unpaired Student ' s t - test ) . ROI , region of interest . K . Top . Representative images of immunofluorescent Ki - 67 staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of Ki - 67 signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0015 , unpaired Student ' s t - test ; D10 vehicle vs D10 GSK - LSD1 p < 0 . 0001 , unpaired Student ' s t - test ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 0012 , Mann - Whitney test ) . ROI , region of interest . L . Top . Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of TUNEL signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * * p < 0 . 001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0009 ; D10 vehicle vs D10 GSK - LSD1 p = 0 . 0001 ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 001 ; unpaired Student ' s t - test with Welch ' s correction ) . ROI , region of interest . M . Representative images of hematoxylin and eosin ( H & E ) staining of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day ( D1 , top ) , 10 days ( D10 , middle ) or until experiment endpoint ( bottom ) . Upper right scale bar represents 50 μm , insert scale bar represents 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Ki-67 staining of PeTa cells treated for 3 days with GSK-LSD1 (1 μM) or ORY-1001 (1 μM). n = 3 biological replicates. Data are represented as means ±SD. ***p < 0.001 (DMSO vs GSK-LSD1 p = 0.0002; DMSO vs ORY-1001 p = 0.0004; unpaired Student's t-test).B. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK-LSD1 or DMSO treatment. C. Quantification of EdU/Propidium iodide staining depicted in Fig 3B. n = 3 biological replicates. Data are represented as means ±SD. **p<0.01; ****p<0.0001; ns, non-significant (G1 phase: DMSO vs GSK-LSD1 p = 0.0059; S phase: DMSO vs GSL-LSD1 p<0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.2338; unpaired Student's t-test). D. Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment. Etoposide serves as positive control for apoptosis, H3 serves as loading control. c.PARP1, cleaved PARP1.E. Caspase-3/7 cleavage activity for PeTa cell line after 6 days of indicated treatment. Etoposide serves as a positive control for apoptosis. n = 3 technical replicates. Data are represented as means ±SD. RLU, relative luminescence units.F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points. H. Representative FACS readout of Annexin V/DAPI staining of PeTa cells after 6 days of 100 nM GSK-LSD1 DMSO treatment. I. Quantification of Annexin V/DAPI staining depicted in Fig 3H. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (Early, Late, Total; unpaired Student's t-test). J. Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK-LSD1 or vehicle treatment. Representative images in Appendix Fig S1B. n = 8, ***p = 0.001 (Vehicle vs GSK-LSD1; unpaired Student's t-test). ROI, region of interest.K. Top. Representative images of immunofluorescent Ki-67 staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of Ki-67 signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, **p<0.01, ****p<0.0001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0015, unpaired Student's t-test; D10 vehicle vs D10 GSK-LSD1 p<0.0001, unpaired Student's t-test; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.0012, Mann-Whitney test). ROI, region of interest.L. Top. Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of TUNEL signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, ***p<0.001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0009; D10 vehicle vs D10 GSK-LSD1 p = 0.0001; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.001; unpaired Student's t-test with Welch's correction). ROI, region of interest.M. Representative images of hematoxylin and eosin (H&E) staining of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day (D1, top), 10 days (D10, middle) or until experiment endpoint (bottom). Upper right scale bar represents 50 μm, insert scale bar represents 10 μm.


Figure 4A . Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK - LSD1 or DMSO treatment . Scale bar , 100 μm . B . Metagene plots showing the average CUT & RUN signal for H3K4me1 , H3K27ac , and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start site ; TES transcription end site . C . ATAC - seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start siteD . Volcano plot showing the - log10 ( adjusted p - value ) and log2 fold - change ( log2FC ) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cell cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor . D . Volcano plot showing the - log10 ( adjusted p - value ) and log2 fold - change ( log2FC ) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cell cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm.B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site.C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start siteD. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor. D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor.


Figure 5B . MA - plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling . Significantly up - and downregulated genes ( FDR ≤ 0 . 05 ; abs ( log2FC ) ≥ 0 . 5 ) are marked in red and blue , respectively . Genes belonging to neuronal differentiation ( GO : 0048666 ) are labelled . FC , fold - change ; CMP , counts per million . H . Gene set enrichment analysis ( GSEA ) of the generated Merkel cell signature on the data in Fig 4B . NES , normalized enrichment score ; FDR , false discovery rate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million.H. Gene set enrichment analysis (GSEA) of the generated Merkel cell signature on the data in Fig 4B. NES, normalized enrichment score; FDR, false discovery rate.


Figure 6A . Schematics and immunoblot of LSD1 co - immunoprecipitation ( coIP ) from PeTa cells . IB , Immunoblot ; MS , mass spectrometry . B . Volcano plot displaying the protein - protein binding partners of LSD1 . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change . C . Protein - protein interaction ( PPI ) mapping of the identified LSD1 binding partners . Individual complexes and p - values are displayed in Fig EV 6A . D . Volcano plot depicting the protein - protein binding partners of LSD1 depleting in PeTa cells treated with ORY - 1001 versus DMSO . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change . E . Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues , ordered according to mean . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system . G . Volcano plot depicting the differentially regulated proteins obtained from Fig 6F . Proteins members of the LSD1 - CoREST complex are labeled and highlighted in red . Proteins significantly upregulated ( - log10 ( p - adjusted ) ≥ 1 ; log2FC ≥ 0 . 75 ) and involved in nervous system development ( GO : 0007399 ) are labelled . FC , fold - change . H . Left . Immunoblot confirmation of HMG20B downregulation . H3 serves as nuclear fraction control and α - Tubulin as loading control . Right . Immunoblot quantification . HMG20B protein levels are normalized to H3 and relative to DMSO control . K . Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B ( shHMG20B ) or negative control ( shRenilla ) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA . Cell survival is depicted at day 8 after transduction and normalized to shRenilla . EV , empty vector control , WT , wild - type ; ΔALPHA , alpha - helices deletion ; ΔCC , coiled - coil domain deletion ; ΔHMG , HMG box deletion .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Schematics and immunoblot of LSD1 co-immunoprecipitation (coIP) from PeTa cells. IB, Immunoblot; MS, mass spectrometry.B. Volcano plot displaying the protein-protein binding partners of LSD1. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. C. Protein-protein interaction (PPI) mapping of the identified LSD1 binding partners. Individual complexes and p-values are displayed in Fig EV 6A. D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change.E. Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues, ordered according to mean. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.G. Volcano plot depicting the differentially regulated proteins obtained from Fig 6F. Proteins members of the LSD1-CoREST complex are labeled and highlighted in red. Proteins significantly upregulated (-log10(p-adjusted)≥1; log2FC≥0.75) and involved in nervous system development (GO:0007399) are labelled. FC, fold-change.H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control.K. Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B (shHMG20B) or negative control (shRenilla) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA. Cell survival is depicted at day 8 after transduction and normalized to shRenilla. EV, empty vector control, WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion.


Figure 7B . RT - qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A . Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control . n = 4 technical replicates . Bar graphs represent mean ± SD . C . Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control . pSMAD1 / 5 / 9 , phospho - SMAD1 / 5 / 9 . D . Immunoblot probing for LSD1 - CoREST complex members at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control . E . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash - out . F . Quantification of EdU / Propidium iodide staining depicted in Fig 7E . n = 3 biological replicates . Data are represented as means ± SD . * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0104 ; S phase : DMSO vs GSK - LSD1 p = 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 0062 ; unpaired Student ' s t - test ) . G . Quantification of FACS Annexin V / DAPI staining of PeTa cells 8 days after drug wash - out . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( unpaired Student ' s t - test ) . I . Subcutaneous tumor growth of pretreated cells ( untreated , n = 8 ; D1 , n = 9 ; D3 , n = 9 ; D6 , n = 4 ) Data are represented as means ± SEM . * p < 0 . 05 , * * * p < 0 . 001 ( untreated vs D1 p = 0 . 023 ; untreated vs D3 p = 0 . 023 ; untreated vs D6 p = 0 . 0007 ; Kruskal - Wallis test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9. D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).


Figure 1C - F . Polysome profiling of yeast cells with ( C - E ) or without ( F ) cycloheximide ( CHX ) treatment , immunoblots for Stm1 and Rpl3 in polysome fractions , and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient - rich condition ( C , F ) , rapamycin treatment for 1 hour ( D ) , or nitrogen starvation for 1 hour ( E ) . Individual polysome profiles were fractionated on a 5 - 50 % sucrose density gradient containing 150 mM KCl into 9 fractions , precipitated and immunoblotted for Stm1 and Rpl3 . Rpl3 is used as a loading control . Each experiment was repeated at least three times .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.


Figure 2G . Polysome profiles of stm1 ∆ cells containing either full - length or N - terminally deleted or C - terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour . H . Binding of full - length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E . 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3 . At least two biological replicates are used for each mutant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.


Figure 3A - B . In vitro kinase assay ( A ) and its quantitation ( B ) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P - γ - ATP . Immunoblots for Stm1 and Raptor are shown . Asterisk indicates the auto - phosphorylation of a mTORC1 component . INK128 is used as an inhibitor of mTOR . Minimum of three replicates are used for statistical analysis by unpaired t - test . Data are presented as the mean ± SD . C . Polysome profiles of wild - type and stm1 ∆ cells under nitrogen starvation for 1 hour . D . Polysome profiles of wild - type and STM1AAA cells under nitrogen starvation for 1 hour . E . Polysome profiles of wild - type and STM1EEE cells under nitrogen starvation for 1 hour . F -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-


Figure 4A - B . Ribosomal subunit profiles ( A ) and quantitation of ribosome content ( B ) of stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells treated with rapamycin for 24 hours . For the ribosomal subunit profile analyses , the cell extracts were treated with 50 mM EDTA and separated on 5 - 25 % sucrose density gradients . At least five biological replicates are used for calculation of p - values from multiple unpaired t - tests are indicated in the graphs . E - G . Immunoblot of Rpl24 - GFP and free GFP ( E ) , quantification of total GFP relative to Pgk1 ( F ) , and free GFP relative to Rpl24 - GFP ( G ) in stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells treated with rapamycin for 24 hours . At least six biological replicates are subjected to statistical analysis using multiple unpaired t - tests . ( s . e . for short exposure ; l . e . for long exposure ) . J - K . Immunoblot ( J ) and the quantification ( K ) of Rpl3 in wild - type and stm1 ∆ cells without rapamycin , or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours . At least six biological replicates are subjected to statistical analysis using multiple unpaired t - tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.


Figure 5A - B . Puromycin incorporation ( A ) and its quantification ( B ) in stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids ( + N ) for 10 , 20 , or 30 minutes . Immunoblots for puromycin , Stm1 , Rpl3 , and Pgk1 are shown . Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t - tests . D - E . Representative fluorescent microscopic images for expression of Rpl24 - GFP ( D ) and its intensity quantification ( E ) in tetrads of control ( RPL24GFP ) and stm1 ∆ RPL24GFP cells ( n = 100 , each ) . Experiments are performed at least three times . Statistical analysis is done by unpaired t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.


Figure 6A - C . Polysome profile and immunoblotting of SERBP1 , RPS6 , and RPL7a across the polysome fractions of HEK293T cells treated with DMSO ( A ) , INK128 ( B ) , or harringtonine ( C ) for 1 hour . Polysome profiles were performed using 5 - 50 % sucrose density gradients containing 150 mM KCl . I - J . Immunoblot for ubiquitin ( I ) and its quantification ( J ) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA ( sgCtrl ) or SERBP1 sgRNA ( sgSERBP1 ) along with INK128 treatment for 48 hours . At least four biological replicates are analyzed by unpaired t - test . Data are presented as the mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.


Figure 1A Control animals ( + / w ; UAS - ralIR / + ) , n = 20 , and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - ralS25N / + ) , n = 20 , and those with salivary gland - specific expression of dominant - negative Ral ( fkh - GAL4 / w ; UAS - ralS25N / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( ral35d / + ) , n = 25 , and ral hypomorph mutants ( ral35d / Y ) , n = 23 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test . G Control animals ( + / w ; UAS - rglIR / + ) , n = 19 , and those with salivary gland - specific knockdown of rgl ( fkh - GAL4 / w ; UAS - rglIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-rglIR/+), n= 19, and those with salivary gland-specific knockdown of rgl (fkh-GAL4/w; UAS-rglIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.


Figure 2A ral hypomorph mutants ( ral35d / Y ; ; UAS - p35 / + ) , n = 20 , animals with salivary gland - specific expression of p35 ( ral35d / + ; ; UAS - p35 / fkh - GAL4 ) , n = 21 , and ral hypomorph mutants with salivary gland - specific expression of p35 ( ral35d / Y ; ; UAS - p35 / fkh - GAL4 ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test comparing the percentages of gland fragments . C Salivary glands dissected from 0 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . D Salivary glands dissected from 13 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . E Quantification of data from ( C and D ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments.C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


Figure 3A ral hypomorph mutants ( ral35d / Y ; ; UAS - Atg6IR / + ) , n = 27 , animals with salivary gland - specific expression of Atg6IR ( ral35d / + ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 20 , and ral hypomorph mutants with salivary gland - specific expression of Atg6IR ( ral35d / Y ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 24 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and ralIR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / UAS - ralIR ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . GFP - marked clone ralIR - expressing cells contain a yellow asterisk . Scale bars represent 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . F Quantification of data from ( E ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ) and are stained for Ref ( 2 ) p ( green ) and DAPI ( blue ) . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . H Quantification of data from ( G ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . I Fat bodies dissected from 4 hour starved animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . J Quantification of data from ( I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2)p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.