C . Top : schemas illustrating the behavioral tasks examined in WT mice . Bottom : the plots represent the discrimination index for the novel object recognition task ( Vehicle N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) , the sociability index ( Vehicle : N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) and the discrimination index for the social discrimination task ( N = 8 for each group ) , measured in each condition . * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index ( exploration of novel object - exploration of familiar object / total object exploration ) equal to zero .

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero.


D . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5 - HT6 - / - mice 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 5 - HT6 - / - mice . They are the means ± S . E . M . of results obtained in four mice per group . n . s . p > 0 . 05 , one - way ANOVA followed by Newman - Keuls test .

O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5-HT6-/- mice 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 5-HT6-/- mice. They are the means ± S.E.M. of results obtained in four mice per group. n.s. p > 0.05, one-way ANOVA followed by Newman-Keuls test.


E . Top : schemas illustrating the behavioral tasks in 5 - HT6 - / - mice . Bottom : the plots represent the discrimination index for the novel object recognition test ( discrimination index : 0 . 30 ± 0 . 05 , N = 10 and 0 . 31 ± 0 . 03 , N = 11 , for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , the 3 - chamber social preference test ( sociability index : 0 . 49 ± 0 . 04 , N = 10 and 0 . 48 ± 0 . 07 , N = 11 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) and the social discrimination test ( discrimination index : 0 . 15 ± 0 . 06 , N = 8 and 0 . 21 ± 0 . 06 , N = 8 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , measured in each condition . One - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line corresponds to a discrimination index equal to zero ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).


A - 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 O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-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.


B . 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 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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).


B . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O

B. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 . 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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

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. 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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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 ,

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O

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

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR

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 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 . ( 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

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR

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. (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 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 . ( 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 O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A. 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 O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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. 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.


( a , b ) Isolated acini from wild - type ( WT ) and Trpml1 - / - mice ( ML1 - / - ) were used to determine the frequency of Ca2 + oscillations in response to stimulation with 3 and 10 pM CCK ( a ) and the response to high concentrations of CCK ( b ) . The average frequency from multiple experiments is given in the columns as mean ± s . e . m in ( a ) . The traces and columns in ( b ) show the mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 cells .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.


( c - e ) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations . ( c ) shows exocytosis during the first 5 min of stimulation , ( d ) shows the two phases , first rapid and second sustained phase , of exocytosis and ( e ) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation . The results are averages of 3 - 4 experiments with each mouse line . denotes p < 0 . 01 or better and # denotes p < 0 . 05 or better .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.


Panel ( a ) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution . Granules size was analyzed in at least 10 images from 3 wild - type and 5 Trpml1 - / - mice .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O

Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice.


( b ) Fixed acini were co - stained with Amylase ( granules ) and LAMP1 ( lysosomes ) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively .

O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively.


( c ) Live wild - type and Trpml1 - / - acini were labeled with mitotracker ( green ) and lysotracker ( red ) . The panels show the merged green , red and DIC images . The large vesicles are marked by white arrows and shown at higher magnification in the inserts . The overlap between lysosomes and granules was determined by ImageJ ( see methods ) . The results are from 14 wild - type and 30 Trpml1 - / - acini each comprising 3 - 7 cells and obtained from 3 wild - type and 4 Trpml1 - / - mice .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

(c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice.


( d ) Co - IP of lysosomal LAMP1 and granules VAMP8 and Rab27b . Note the increased lysosomal / granules interaction .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.


( e ) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . The results are the mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type and 3 Trpml1 - / - mice .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O

(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.


( a , b ) Plasmaamylase ( a ) and pancreaticMPO ( b ) in 3 mice of each line .

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

(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line.


( c ) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H & amp ; amp ; E staining . Shown are examples of images and the average damage in the columns , which show the mean ± s . e . m of 4 experiments with each mouse line .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.


( d ) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min . Shown are example images and the averages .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.


( e ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide . The figure shows example images before stimulation and the averaged fluorescence intensity .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.


( f ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3 . The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as the mean ± s . e . m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.


( a ) Isolated acini from wild - type ( black ) and Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . The traces are the mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.


( b , c ) Saliva was collected from wild - type ( black ) and Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like and the results are given as the mean ± s . e . m .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O 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, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.


( d ) the saliva collected in ( c ) was used to determine the amount of secreted amylase .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(d) the saliva collected in (c) was used to determine the amount of secreted amylase.


( e , f ) Isolated acini from the parotid glands of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times ( e ) and the indicated isoproterenol concentrations during 30 min stimulation ( f ) . The results are the mean ± s . e . m obtained from two mice of each line .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


( a ) shows example TEM images and ( b ) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild - type mice and 14 images obtained from 4 Trpml1 - / - mice .

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O

(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.


( c ) Co - IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b . Input control is actin .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.


( d ) Acini isolated from the parotid glands of wild - type and Trpml1 - / - mice were incubated with mitotracker ( green ) and lysotracker . The large vesicles in the secretory granules / lysosomal area are marked by white arrows . The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.


( e ) Shown is the time course ( left panel ) and averaged 10 min secretion ( columns ) of the lysosomal marker acid phosphatase in the saliva collected from wild - type ( black ) and Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol . The mean ± s . e . m is from 4 mice of each line .

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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


( a , b ) example traces of mEPSCs recorded from wild - type ( a ) and Trpml1 - / - ( b ) micebrain slices as detailed in Methods . The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of the events at each mEPSC . The lower columns show the distribution of the higher amplitude mEPSCs . ( d ) the frequency of mEPSCs in each line . The results in ( c , b ) are the mean ± s . e . m of 8 experiments each obtained from 4 mice of each line .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.


( e ) the mean ± s . e . m of the Ca2 + signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild - type and Trpml1 - / - slices , respectively .

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(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


( a ) example TEM images of synapses in wild - type and Trpml1 - / - micebraincortex . ( b ) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) example TEM images at low magnification of wild - type and Trpml1 - / - micebraincortical slices with the dark PSD profiles marked by white arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 images obtained from 3 mice of each line .

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(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.


( e ) Glutamate levels and secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) and Trpml1 - / - ( green ) neurons and neurons stimulated by depolarization with 30 mM KCl ( blue and red ) .

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(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).


( f ) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) and Trpml1 - / - neurons transfected with TRPML1 ( black ) and untransfected ( red ) and TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl .

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(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.


( g ) averaged Co - IP of SNAP25 and VAMP2 in three brain regions of wild - type and Trpml1 - / - mice . The images of all blots are given in Extended view Figure E4 .

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(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


( B ) Assessment of exogenous DDX60 expression . HEK293T cells transfected with DDX60 wild type ( wt ) , or DDX60 mutants and analyzed by western blot for DDX60 , ß - actin and GAPDH ( loading controls ) , and RFP ( reporter ) . DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below .

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(B) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.


( C ) HCV antiviral assays with DDX60 wt or mutant panel . Huh - 7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase ( Fluc , negative , and transfection control ) , IRF1 ( positive antiviral control ) , DDX60 wt , or DDX60 mutants and infected with HCV - Ypet , a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C , E1 , E2 , p7 , NS2 , NS3 , 4A , 4B , NS5A , and NS5B is driven by EMCV IRES . Data information : For ( C ) percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Data information: For (C) percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( D ) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs . Huh - 7 cells transfected as in ( C ) and infected with either bicistronic HCV - Ypet ( left ) or monocistronic HCV J6 / JFH - 5AB - YPet . Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B . Data information : For ( D ) , percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc .

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(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. Data information: For (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( A ) Plasmid - based dual luciferase bicistronic reporter assays . HEK293T cells co - transfected with dual luciferase bicistronic reporter plasmid ( Renilla luciferase ( Rluc ) translationally driven by a 5 ' cap , and Firefly luciferase ( Fluc ) translationally driven by different IRESs as indicated ) and GFP plasmid ( negative control ) or increasing amounts of IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A mutant . Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid . Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells ( left ) and ratio of IRES Fluc units over 5 ' cap Rluc units ( middle ) . Diagram to the right of ratios plot depicts expected ratios of IRES - driven Fluc units to 5 ' cap - driven Rluc units given either : equal translation of 5 ' cap - driven Rluc and IRES - driven Fluc ( top ) , greater translation of IRES - driven Fluc ( middle ) , or greater translation of 5 ' cap - driven Rluc ( bottom ) . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against GFP .

O O O 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-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5' cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5' cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5' cap-driven Rluc units given either: equal translation of 5' cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5' cap-driven Rluc (bottom). Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( B ) RNA - based monocistronic luciferase reporter assays . HEK293T cells transfected with GFP ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and subsequently transfected with in vitro transcribed 5 ' cap or different IRES driven Fluc mRNA constructs as indicated . Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against GFP .

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(B) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5' cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( A - E ) ( A , B , E ) Multi - cycle infection assays with type I and type II IRES containing viruses . HeLa cells stably expressing Firefly luciferase ( Fluc ) ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and infected with ( A ) encephalomyocarditis virus , ( B ) poliovirus , or ( E ) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV ( EMCV - IRES - PV ) at MOI 0 . 001 . Supernatants were collected 24 hours post infection ( hpi ) ( EMCV ) or 48 hpi ( poliovirus and EMCV - IRES - PV ) and titers determined via plaque assay on HeLa cells . ( C , D ) Single - cycle infection assays with type I and type II IRES containing viruses . BHK - J cells stably expressing empty vector ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and infected with ( C ) foot and mouth disease virus , or ( D ) bovine enterovirus - 1 at MOI 1 . Supernatants were collected 5 hours post infection ( hpi ) and titers determined via plaque assay on BHK - 21 clone 13 cells . Data information : Data shows mean ± SEM percent infectious titers relative to Fluc from at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against DDX60 E890A .

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(A-E) (A, B, E) Multi-cycle infection assays with type I and type II IRES containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) encephalomyocarditis virus, (B) poliovirus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV-IRES-PV) at MOI 0.001. Supernatants were collected 24 hours post infection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV-IRES-PV) and titers determined via plaque assay on HeLa cells. (C, D) Single-cycle infection assays with type I and type II IRES containing viruses. BHK-J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) foot and mouth disease virus, or (D) bovine enterovirus-1 at MOI 1. Supernatants were collected 5 hours post infection (hpi) and titers determined via plaque assay on BHK-21 clone 13 cells. Data information: Data shows mean ± SEM percent infectious titers relative to Fluc from at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against DDX60 E890A.


B ) HEK293T cells transfected with DDX60 wt or DDX60 E890A ( negative control ) and transfected with in vitro transcribed 5 ' cap - or IRES - driven Fluc mRNA constructs as indicated and lysed 16 hours later . ( B ) IRES - or cap - driven translation from the same samples assayed in parallel using luciferase assay . Data information : B ) Mean ± SD from at least n = 3 biological replicates ; * * * * FDR < 0 . 01 % ( p < 0 . 0001 ) using unpaired t - test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells . ns , non - significant .

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B) HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5' cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 16 hours later. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. Data information: B) Mean ± SD from at least n=3 biological replicates; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells. ns, non-significant.


( C , HEK293T cells stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected . Transfected cells were then treated with either PBS ( negative control ) or transfected with LMW poly ( I : C ) . In parallel , untransfected cells were treated with either PBS ( mock ) , transfected with LMW poly ( I : C ) , or treated with IFN - ß ( positive control ) . Cells were subsequently used for a luciferase assay to assess ISRE activity ( C ) Data information : ( C ) Mean ± SD from at least n = 3 biological replicates ; * p < 0 . 05 using unpaired t - test with Welch ' s correction , ns , not significant using repeated measures one - way ANOVA with Geisser - Greenhouse correction comparing RIG - I transfected vs RIG - I + DDX60 wt or RIG - I + DDX60 E890A transfected cells .

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(C, HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (C) Data information: (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60 wt or RIG-I + DDX60 E890A transfected cells.


D ) HEK293T cells stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected . western blot for analysis of DDX60 , RIG - I , ß - actin ( loading control ) , or GAPDH ( loading control ) protein products ( D ) . Quantification of DDX60 and RIG - I band intensities relative to ß - actin are shown below each respective lane in ( D ) . Data information : ( D ) representative western blot from ( C ) .

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D) HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (D). Quantification of DDX60 and RIG-I band intensities relative to ß-actin are shown below each respective lane in (D). Data information: (D) representative western blot from (C).


( B ) Effect of DDX60 on 5 ' cap ( left ) or EMCV IRES ( right ) driven Fluc mRNA polysomes . Data information : Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions , from n = 3 biological replicates .

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(B) Effect of DDX60 on 5' cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes. Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n=3 biological replicates.


( B ) Cells depicted in ( A ) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors , applying cell lysates to 15 % - 50 % sucrose gradients , and subjecting to ultracentrifugation . Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance ( 254 nm ) .

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(B) Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).


( C ) Representative results of RNA electrophoresis analysis of individual fractions . Shown are results from one replicate of DDX60 - transfected , EMCV - IRES - PV - infected cells . RNA was analyzed by Bioanalyzer to visualize 28S , and 18S ribosome subunit distribution . RIN , RNA integrity number .

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(C) Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity number.


( D ) Effect of DDX60 expression on parental poliovirus ( type I IRES , purple circles ) versus chimeric poliovirus ( type II IRES , red squares ) VP4 mRNA polysomes ( left ) and cellular GAPDH mRNA polysomes in the same samples . Data information : Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n = 3 replicate plates per condition .

O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

(D) Effect of DDX60 expression on parental poliovirus (type I IRES, purple circles) versus chimeric poliovirus (type II IRES, red squares) VP4 mRNA polysomes (left) and cellular GAPDH mRNA polysomes in the same samples. Data information: Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n=3 replicate plates per condition.


B Expression ( cells ) and secretion ( media ) of VgrG1 from V . parahaemolyticus RIMD 2210633 derivative POR1 ( Vpara ; T6SS1 + ) and its T6SS1 - mutant ( Vpara / ∆ hcp1 ) , and from V . natriegens ( Vnat ) carrying the indicated plasmids . Samples were treated ( + ) or not ( - ) with 20 µM phenamil to activate surface sensing in media containing 3 % NaCl at 30 ° C for 5 h . Loading control ( LC ) is shown for total protein lysates . Arrows denote bands corresponding to VgrG1 . Asterisks denote non - specific bands detected in Vnat samples .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O

B Expression (cells) and secretion (media) of VgrG1 from V. parahaemolyticus RIMD 2210633 derivative POR1 (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from V. natriegens (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in Vnat samples.


C Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with the indicated attackers , as described in B , on media containing 3 % NaCl at 30 ° C . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above . A significant difference was considered as P < 0 . 05 . DL , assay detection limit .

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

C Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit.


B Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with wild type V . natriegens attackers harboring the indicated plasmids , on media containing 3 % NaCl at 30 ° C . Data information : data are shown as the mean ± SD , n = 3 technical replicates ; statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens attackers harboring the indicated plasmids, on media containing 3% NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C Expression ( cells ) and secretion ( media ) of VgrG1 from V . natriegens containing an arabinose - inducible vp1407 in the chromosomal dns locus ( VnatReg ) and harboring an empty plasmid ( pEmpty ) or plasmids containing VpT6SS1 that lacks vp1407 ( pT6SS1Ind ) or both vp1407 and hcp1 ( pT6SS1Ind / ∆ hcp1 ; T6SS - ) . Samples were grown in media containing 3 % NaCl and either supplemented ( + ) or not ( - ) with 0 . 1 % arabinose ( Ara ) at 30 ° C . Loading control ( LC ) is shown for total protein lysates .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.


E Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers carrying the indicated plasmids on solid media plates , as described in C . Data information : data are shown as the mean ± SD , n = 3 technical replicates ; statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

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

E Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A , B Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 or 24 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose ( A ) , or on media containing 0 . 1 % arabinose at 28 ° C ( B ) . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C - F Viability counts of the indicated prey bacteria before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose at 28 ° C . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

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

C-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


Viability counts of V . natriegens ( cyan ) , V . vulnificus ( purple ) , A . jandaei ∆ tssB ( brown ) , E . coli ( green ) , and S . enterica ( gray ) prey before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers ( yellow ) harboring the indicated plasmids , on media containing 3 % NaCl at 28 ° C . The left panels show prey survival when competing alone ( single prey ) against the attacker at a 4 : 1 ( attacker : prey ) ratio ; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10 : 1 : 1 : 1 : 1 : 1 ( attacker : prey ) ratio . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Viability counts of V. natriegens (cyan), V. vulnificus (purple), A. jandaei ∆tssB (brown), E. coli (green), and S. enterica (gray) prey before (0 h) and after (4 h) co-incubation with VnatReg attackers (yellow) harboring the indicated plasmids, on media containing 3% NaCl at 28 °C. The left panels show prey survival when competing alone (single prey) against the attacker at a 4:1 (attacker:prey) ratio; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10:1:1:1:1:1 (attacker:prey) ratio. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A - D ( A , B , D ) Viability counts of V . parahaemolyticus RIMD 2210633 ∆ hcp1 ( A ) , V . alginolyticus 12G01 ∆ hcp1 / ∆ hcp2 ( B ) , and V . natriegens ( D ) prey bacteria before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose at 28 ° C . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above . A significant difference was considered as P < 0 . 05 . ( C ) Illustration summarizing the competitions shown in ( A , B ) . Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless ( yellow ) are colored to match the bacterium from which they were derived .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D (A, B, D) Viability counts of V. parahaemolyticus RIMD 2210633 ∆hcp1 (A), V. alginolyticus 12G01 ∆hcp1/∆hcp2 (B), and V. natriegens (D) prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. ( C) Illustration summarizing the competitions shown in (A, B). Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless (yellow) are colored to match the bacterium from which they were derived.


( A ) HEK GFP - LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved ( large circles ) or not starved ( small circles ) for 2 h , followed by fixation and counterstaining of the nuclei by Hoechst . The images were processed for high - content image analysis to quantify the number of GFP - LC3 spots per cell . The graph shows the average of

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O

(A) HEK GFP-LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved (large circles) or not starved (small circles) for 2 h, followed by fixation and counterstaining of the nuclei by Hoechst. The images were processed for high-content image analysis to quantify the number of GFP-LC3 spots per cell. The graph shows the average of three


( B ) Selected PX domain proteins were depleted by transfection of HEK GFP - LC3 with the four individual siRNA oligos of the pools used in A . The cells were treated , imaged , and analyzed as in A . The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR . The graph shows the average of two independent experiments in triplicate with a total number of 18 , 000 cells . Coloring is as in A .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Selected PX domain proteins were depleted by transfection of HEK GFP-LC3 with the four individual siRNA oligos of the pools used in A. The cells were treated, imaged, and analyzed as in A. The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR. The graph shows the average of two independent experiments in triplicate with a total number of 18,000 cells. Coloring is as in A.


( C ) Micrographs of starved HEK GFP - LC3 cells treated with nontargeting or siSNX18 - 3 siRNA . ( left and middle ) 3 × 3 fields of cells . ( right ) Close - up of a few cells from the respective population . The images in the right panels are shown again in Fig . S1 B alongside cells treated with other SNX18 - targeting siRNAs . Bars , 10 µm . See also Table S1 and Table S2 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(C) Micrographs of starved HEK GFP-LC3 cells treated with nontargeting or siSNX18-3 siRNA. (left and middle) 3 × 3 fields of cells. (right) Close-up of a few cells from the respective population. The images in the right panels are shown again in Fig. S1 B alongside cells treated with other SNX18-targeting siRNAs. Bars, 10 µm. See also Table S1 and Table S2.


( A ) HEK GFP - LC3 cells transfected with control ( siCtrl ) or two different SNX18 siRNA oligos ( siSNX18 - 3 and siSNX18 - 5 ) were starved or not starved for 2 h in the presence or absence of BafA1 . GFP - LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting . The graph shows the average GFP - LC3 - II relative to actin normalized to siCtrl - 1 fed ± SEM ( error bars ) , n = 4 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(A) HEK GFP-LC3 cells transfected with control (siCtrl) or two different SNX18 siRNA oligos (siSNX18-3 and siSNX18-5) were starved or not starved for 2 h in the presence or absence of BafA1. GFP-LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting. The graph shows the average GFP-LC3-II relative to actin normalized to siCtrl-1 fed ± SEM (error bars), n = 4.


( B ) The degradation of long - lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3 - MA and normalized to the degradation in fed cells ( mean ± SEM [ error bars ] , n = 3 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) The degradation of long-lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3-MA and normalized to the degradation in fed cells (mean ± SEM [error bars], n = 3).


( C ) HEK GFP - LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA - resistant myc - SNX18 plasmid . The number of GFP - LC3 spots per cell was quantified ( graph shows mean ± SEM [ error bars ] , n = 3 ) .

O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(C) HEK GFP-LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA-resistant myc-SNX18 plasmid. The number of GFP-LC3 spots per cell was quantified (graph shows mean ± SEM [error bars], n = 3).


( D ) HEK GFP - LC3 cells were transfected with myc - SNX18 or a myc control plasmid and treated as in A . The graph shows the average LC3 - II levels relative to actin normalized to myc - transfected fed cells , ± SEM ( error bars ) . n = 3 .

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(D) HEK GFP-LC3 cells were transfected with myc-SNX18 or a myc control plasmid and treated as in A. The graph shows the average LC3-II levels relative to actin normalized to myc-transfected fed cells, ±SEM (error bars). n = 3.


( E ) HEK GFP - LC3 cells were transfected with control or Atg7 siRNA and then with myc - SNX18 or a myc control plasmid , followed by quantification of the number of GFP - LC3 spots per cell ( graphs show mean ± SEM [ error bars ] , n = 3 ) . Representative images are shown . Bars , 5 µm .

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(E) HEK GFP-LC3 cells were transfected with control or Atg7 siRNA and then with myc-SNX18 or a myc control plasmid, followed by quantification of the number of GFP-LC3 spots per cell (graphs show mean ± SEM [error bars], n = 3). Representative images are shown. Bars, 5 µm.


( F ) The degradation of long - lived protein in HeLa cells transfected with myc - SNX18 or a myc control plasmid was quantified as in B ( graph shows mean ± SEM [ error bars ] , n = 4 ) . * , P < 0 . 05 ; * * , P < 0 . 01 . See also Figs . S1 and S2 .

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(F) The degradation of long-lived protein in HeLa cells transfected with myc-SNX18 or a myc control plasmid was quantified as in B (graph shows mean ± SEM [error bars], n = 4). *, P < 0.05; **, P < 0.01. See also Figs. S1 and S2.


( A ) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting . Migration of different organelles is indicated , based on the positions in the gradient of calnexin ( ER ) , GM130 ( Golgi stack / CGN ) , SNX9 ( plasma membrane ) , AP - 1 , TfR and Rab11a ( Endosomes / TGN ) , and LC3 - II and GABARAP - II ( autophagosomes ) .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O

(A) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting. Migration of different organelles is indicated, based on the positions in the gradient of calnexin (ER), GM130 (Golgi stack/CGN), SNX9 (plasma membrane), AP-1, TfR and Rab11a (Endosomes/TGN), and LC3-II and GABARAP-II (autophagosomes).


( B ) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130 . Panels to the right show enlarged views of the boxed regions .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

(B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions.


( C ) HeLa cells were transfected with GFP - Atg14L and immunostained for SNX18 and calnexin . The boxed regions are enlarged on the top and bottom .

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(C) HeLa cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom.


( D and E ) HEK GFP - DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 ( D ) or WIPI2 ( E ) . Results shown are mean ± SEM ( error bars ) , n = 3 with 30 cells in each experiment . * , P < 0 . 05 .

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(D and E) HEK GFP-DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 (D) or WIPI2 (E). Results shown are mean ± SEM (error bars), n = 3 with 30 cells in each experiment. *, P < 0.05.


( F ) HeLa cells were transfected with control or SNX18 siRNA , starved for 2 h , and immunostained against SNX18 and Atg16L1 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O

(F) HeLa cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1.


( G ) HEK GFP - DFCP1 cells were transfected with control or SNX18 siRNA , starved for 50 min , and immunostained for Atg16L1 . All images are from confocal microscopy . Inset panels show enlarged views of the boxed regions . Bars , 5 µm . See also Fig . S3 .

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(G) HEK GFP-DFCP1 cells were transfected with control or SNX18 siRNA, starved for 50 min, and immunostained for Atg16L1. All images are from confocal microscopy. Inset panels show enlarged views of the boxed regions. Bars, 5 µm. See also Fig. S3.


( A ) HeLa GFP - LC3 cells were transfected to express myc - SNX18 , immunostained against myc , and analyzed by confocal imaging . Asterisks indicate a nontransfected cell . Bars : ( main panels ) 20 µm ; ( inset ) 10 µm .

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(A) HeLa GFP-LC3 cells were transfected to express myc-SNX18, immunostained against myc, and analyzed by confocal imaging. Asterisks indicate a nontransfected cell. Bars: (main panels) 20 µm; (inset) 10 µm.


( B ) HeLa cells transfected with flag - Atg16L1 and myc - SNX18 were starved or not starved for 2 h before immunostaining with anti - myc and anti - flag antibodies and confocal microscopy . Bars , 5 µm .

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(B) HeLa cells transfected with flag-Atg16L1 and myc-SNX18 were starved or not starved for 2 h before immunostaining with anti-myc and anti-flag antibodies and confocal microscopy. Bars, 5 µm.


( D ) HeLa cells were transfected with the indicated myc - SNX18 plasmids for 6 h and starved for an additional 2 h before harvest . Equal parts of cytosol ( C ) and membrane ( M ) fractions were analyzed by Western blotting . The graph shows the average membrane - bound myc - SNX18 ± SEM ( error bars ) , n = 3 .

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(D) HeLa cells were transfected with the indicated myc-SNX18 plasmids for 6 h and starved for an additional 2 h before harvest. Equal parts of cytosol (C) and membrane (M) fractions were analyzed by Western blotting. The graph shows the average membrane-bound myc-SNX18 ± SEM (error bars), n = 3.


( E and F ) HEK GFP - LC3 cells were transfected with the indicated mCherry - SNX18 ( E ) or mycSNX18 ( F ) constructs for 16 h . The number of GFP - LC3 spots per transfected cell ( E ; graph shows mean ± SEM [ error bars ] , n = 5 ) or ratio of LC3 - II to actin determined by Western blotting ( F ) was quantified .

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(E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified.