Figure 2A Representative kymographs of LysoTracker - labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA ( shSc ) , LAMTOR2 shRNA ( shLAMTOR2 ) , or Raptor shRNA ( shRaptor ) . Scale bar , 5 µm . B Quantitative analysis of lysosomal movement from kymographs ( N = 44 , 24 , and 19 neurons for shSc , shLAMTOR2 , and shRaptor , respectively , from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Sidak ' s post - test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant . C , D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons . C Representative images of a neuron at 0 and 4 min after photobleaching . D Quantification of the lysosomes in C undergoing retrograde ( red ) or anterograde ( cyan ) transport ( N = 7 and 8 neurons for shSc and shRaptor , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Mann - Whitney U test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant . E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594 - conjugated transferrin ( red , Tf 594 ) in dendrites . Shown are dendritic segments ( upper ) and kymographs ( lower ) . Scale bar , 5 µm . F Quantitative analysis of vesicular movement from kymographs ( N = 10 neurons from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Mann - Whitney U test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

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

Figure 2A Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.C, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. C Representative images of a neuron at 0 and 4 min after photobleaching. D Quantification of the lysosomes in C undergoing retrograde (red) or anterograde (cyan) transport (N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.


Figure 3A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA ( shLAMTOR1 ) or scrambled shRNA ( shSc ) and Accell lyspersin siRNA or control siRNA . Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites . N = 17 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± SEM . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - test . C - F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction . C , E , F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc ; they were treated with vehicle control or ML - SI1 ( 20 µM , C , n = 16 - 49 neurons from 3 - 10 independent experiments ) , or ML - SA1 ( 20 µM , E , n = 13 - 49 neurons from 3 - 10 independent experiments ) , or CilioD ( 20 µM , F , n = 12 - 49 neurons from 3 - 10 independent experiments ) , and imaged with LysoTracker to visualize lysosomal trafficking in dendrites . D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA ; they were imaged as described above . N = 7 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± SEM . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - test .

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Figure 3A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.C-F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. C, E, F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML-SI1 (20 µM, C, n = 16-49 neurons from 3-10 independent experiments), or ML-SA1 (20 µM, E, n = 13-49 neurons from 3-10 independent experiments), or CilioD (20 µM, F, n = 12-49 neurons from 3-10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.


Figure 4F Quantification of co - IP results . N = 3 independent experiments . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test ( F ) . * P < 0 . 05 , * * * P < 0 . 001 .

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Figure 4F Quantification of co-IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test (F). *P < 0.05, ***P < 0.001.


Figure 5A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus . Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT - 2031 peptide . Wes protein analysis with anti - LAMTOR1 and - TRPML1 antibodies of immunoprecipitation performed with anti - LAMTOR1 antibodies or negative control anti - HA antibodies using whole hippocampal homogenates from naïve , TAT or TAT - 2031 - treated mice . B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve , TAT or TAT - 2031 - treated mice . N = 3 mice for each group . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test * * P < 0 . 01 , * * * P < 0 . 001 . C Representative images from proximity ligation assay ( PLA ) performed on brain slices from naïve , TAT or TAT - 2031 - treated mice . Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 μm . D Quantification of the number of PLA signals in CA1 from naïve , TAT or TAT - 2031 - treated mice . N = 6 mice for each group . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 5A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1 and -TRPML1 antibodies of immunoprecipitation performed with anti-LAMTOR1 antibodies or negative control anti-HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT-2031-treated mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.C Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT-2031-treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.


Figure 6D TAT - 2031 treatment ( 10 µM ) increased ML - SA1 - induced TRPML1 Ca2 + release in both control and LAMTOR1 KD cells . Data information : traces represent the mean values of each group . G Treatment with GPN ( 200 µM ) , BAPTA - AM ( 20 µM ) , or ML - SI1 ( 20 µM ) blocked ML - SA1 - induced TRPML1 - GCaMP6m responses in neurons . Data information : traces represent the mean values of each group . I Both LAMTOR1 KD and TAT - 2031 treatment ( 10 µM ) increased ML - SA1 - induced TRPML1 - GCaMP6m responses in neurons . J Quantification of peak responses as shown in I . N = 6 - 13 cells from 3 independent experiments . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Tukey ' s post hoc analysis * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # # # P < 0 . 001 , n . s . , not significant . traces represent the mean values of each group . K LAMTOR1 KD increased PI ( 3 , 5 ) P2 ( 0 . 5 µM ) - induced TRPML1 - GCaMP6m responses and the blocking effect of ML - SI1 treatment ( 20 µM ) . L Quantification of peak TRPML1 - GCaMP6m responses as shown in K . N = 6 - 16 cells from 3 independent experiments . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Tukey ' s post hoc analysis * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # # # P < 0 . 001 , n . s . , not significant . traces represent the mean values of each group .

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

Figure 6D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells. Data information: traces represent the mean values of each group.G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group.I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.K LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)-induced TRPML1-GCaMP6m responses and the blocking effect of ML-SI1 treatment (20 µM). L Quantification of peak TRPML1-GCaMP6m responses as shown in K. N = 6-16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.


Figure 7A - D Effects of LAMTOR1 KD and ML - SI1 treatment on TBS - induced LTP ( A ) or LFS - induced LTD ( C ) in CA1 . B , D Means ± SEM of fEPSPs measured 40 min after TBS ( B , n = 4 - 5 slices from 4 - 5 mice ) or LFS ( D , n = 4 - 8 slices from 4 - 8 mice ) in different groups . Data information : Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two - way ANOVA with Tukey ' s post - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 . Insets show representative traces of evoked fEPSPs before and 40 min after TBS / LFS . Scale bar 0 . 5 mV / 10 ms . G - J Effects of TAT - 2031 treatment on TBS - induced LTP ( G ) or LFS - induced LTD ( I ) in CA1 . H , J Means ± SEM of fEPSPs measured 40 min after TBS ( H , n = 6 slices from 6 mice ) or LFS ( J , n = 3 - 4 slices from 3 - 4 mice ) in different groups . Data information : Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc , Vehicle , or TAT Insets show representative traces of evoked fEPSPs before and 40 min after TBS / LFS . Scale bar 0 . 5 mV / 10 ms . K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor , FK506 , on LFS - induced LTD in CA1 . L Means ± SEM of fEPSPs measured 45 min after LFS in different groups ( n = 3 - 4 slices from 3 - 4 mice ) . Data information : Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two - way ANOVA with Tukey ' s post - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc , Vehicle , or TAT , # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 . Insets show representative traces of evoked fEPSPs before and 40 min after LFS . Scale bar 0 . 5 mV / 10 ms .

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

Figure 7A-D Effects of LAMTOR1 KD and ML-SI1 treatment on TBS-induced LTP (A) or LFS-induced LTD (C) in CA1. B, D Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4-5 slices from 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.G-J Effects of TAT-2031 treatment on TBS-induced LTP (G) or LFS-induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3-4 slices from 3-4 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS-induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms.


Figure 8LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p - GluA1 to GluA1 in hippocampal neurons , and this effect was reversed by ML - SI1 treatment . A Representative images of CA1 pyramidal neurons stained with anti - p - GluA1 S845 ( magenta ) , anti - GluA1 ( red ) , and anti - GFP ( green ) antibodies . Scale bar = 20 µm . E Representative images of proximal dendrites of hippocampal neurons stained with anti - GluA1 ( green ) , anti - LAMP2 ( red ) , and anti - GFP ( gray ) antibodies . Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false - colored green and GFP false - colored gray to better show colocalization of GluA1 with LAMP2 . Arrowheads indicate clearly co - localized puncta . Scale bar , 5 µm .

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Figure 8LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm.E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm.


Figure 9A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one . LAMTOR1 shRNA - injected mice exhibited a significant deficit 24 h after training , which was reversed by ML - SI1 treatment ( N = 7 - 18 mice ) . B % freezing for different experimental groups in context memory ( N = 7 - 19 mice ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Tukey ' s post - test . * P < 0 . 05 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 .

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Figure 9A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1.


f1 ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( a ) The cell viability was assayed by trypan blue staining . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) . ( b ) Cell count and viability assay using Muse ( Millipore , USA ) . Data are mean from three independent experiments , with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) . ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 µM ecdysone for 24 h . ( c ) Caspase activity was measured from total cell lysate on DEVD - AMC , represented as fold increase relative to control RNAi . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) . ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( d ) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49 . Relative dUTX expression levels following knockdown using the two independent dsRNA are shown ( n = 3 ) with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) .

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

f1(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student's t-test).(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student's t-test).


f2 ( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( a ) GST and GST - dUTX - NR containing the nuclear hormone domain were used in pull - down experiments with 35S - labelled EcR and Usp in the presence of ethanol control ( − ) or 10 μM ecdysone ( + ) . The GST input proteins are shown in Coomassie Brilliant blue - stained gel , GST is labelled with arrow head and dUTX - NR with an asterisk . The EcR and Usp inputs are shown labelled with arrows . ( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( b ) Immunoprecipitation ( IP ) of EcR and dUTX in SL2 cells . Cells were transfected with HA - dUTX and the cell lysates were immunoprecipitated using anti - HA or a control IgG isotype antibody . The immunoprecipitates were analysed by western blotting using the anti - EcR and anti - HA antibodies . ( c ) Colocalization of dUTX and EcR on salivary gland polytene chromosomes . Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG - dUTX with dUTX ( green ) and EcR ( red ) , with Hoechst - stained DNA ( blue ) . Arrows indicate examples of colocalized bands . No signal was detected when immunostaining with secondary antibody only . Scale bar represent 5 μm .

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f2(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows.(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies.(c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.


f3 ( a ) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF . Ovals indicate the position of salivary glands and fragments . Scale bar represent 50 μm . Quantification of the salivary gland phenotypic data was at 24 h RPF . ( b , c ) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF . ( b ) Cleaved caspase - 3 antibody ( green ) and Lamin antibody ( red ) staining are shown in control and dUTX1 salivary glands at 14 h RPF . Scale bar represents 50 μm . ( c ) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD - AMC as a substrate . Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts . The error bars represent s . e . m . * P - value0 . 05 ( Student ' s t - test ) . ( d ) Autophagy puncta examined using mCherry - Atg8a ( red ) and nuclei stained with Hoechst ( blue ) . Scale bar represents 100 μm . The quantitation of the mCherry - Atg8a puncta as the mean fluorescent pixel intensity per cell ( control n = 242 , dUTX1 n = 224 ) , with error bars representing s . e . m . * P - value0 . 05 ( Student ' s t - test ) .

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f3(a) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF.(b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student's t-test).(d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student's t-test).


f4 ( a ) Levels of dark , dronc , drice and rpr transcripts were measured from third instar larvae ( L3 ) , 6 h and 14 h RPF salivary glands from control ( blue ) and dUTX1 ( red ) by qPCR and expressed relative to the internal control gene rp49 . ( b ) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control ( blue ) and dUTX1 ( red ) were analysed by qPCR at L3 , 6 h and 14 h RPF . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

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f4(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49.(b) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control (blue) and dUTX1 (red) were analysed by qPCR at L3, 6 h and 14 h RPF. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


f5 ( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of dark , dronc , drice and rpr ( a ) ( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of Atg1 , Atg2 , Atg3 , Atg4 , Atg5 , Atg6 , Atg7 , Atg8a , Atg9 , Atg12 and Atg18 ( b ) , expressed relative to the internal control gene Gapdh . Results are shown as the fold enrichment of % input compared with IgG control . ( c ) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP . SL2 cells knocked down for dUTX were chromatin - immunoprecipitated with anti - H3K27me3 and anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with RNAi control . ( d ) The occupancy of EcR on the promoter regions of dark , dronc and Atg1 by ChIP expressed relative to the internal control gene rp49 . Chromatin was immunoprecipitated with anti - EcR - B1 and anti - GFP ( IgG control ) antibodies from SL2 cell following ecdysone treatment . Results are shown as the fold enrichment of % input compared with IgG control . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

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f5(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a)(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control.(c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control.(d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


f6 ( a ) Levels of BR - C and E93 transcripts were measured in salivary glands isolated from third instar larvae ( L3 ) , 6 h and 14 h RPF control ( blue line ) and dUTX1 ( red line ) using qPCR and expressed relative to the internal control gene rp49 . ( b ) The occupancy of dUTX on the promoter regions of BR - C and E93 genes detected by ChIP . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with IgG control . In the right hand panel abundance of H3K27me3 on the promoters of BR - C and E93 genes as detected by ChIP is shown . SL2 cells knockdown for dUTX ( dUTX RNAi ) were chromatin - immunoprecipitated with anti - H3K27me3 and rabbit IgG ( control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with RNAi control . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Students t - test ) .

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

f6(a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49.(b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).


f7 ( a ) Mosaic clones of dUTX1 ( hsFLP ; ubi - GFP FRT40A / dUTX1 FRT40A ) from third instar larval salivary glands with wild - type cells marked by nuclear - localized GFP ( green ) . dUTX1 clones have high levels of H3K27me3 ( red ) compared with the neighbouring control cells marked by GFP . Scale bar represents 50 μm . ( b ) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands . Alpha - tubulin and H3 serve as loading controls . ( c ) Expression of dUTX wild - type ( dUTXWT ) and dUTX catalytic mutant ( dUTXJmj ) in the salivary glands ( using Sgs3 - GAL4 driver ) of dUTX1 . Histology analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1 ; dUTXJmj * compared with control and dUTX1 ; dUTXWT at 24 h RPF . Ovals indicate the position of salivary glands and fragments . Scale bar represent 50 μm . Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right .

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f7(a) Mosaic clones of dUTX1 (hsFLP; ubi-GFP FRT40A/dUTX1 FRT40A) from third instar larval salivary glands with wild-type cells marked by nuclear-localized GFP (green). dUTX1 clones have high levels of H3K27me3 (red) compared with the neighbouring control cells marked by GFP. Scale bar represents 50 μm.(b) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands. Alpha-tubulin and H3 serve as loading controls.(c) Expression of dUTX wild-type (dUTXWT) and dUTX catalytic mutant (dUTXJmj) in the salivary glands (using Sgs3-GAL4 driver) of dUTX1. Histology analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1; dUTXJmj* compared with control and dUTX1; dUTXWT at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right.


Figure 2HEp - 2 cells remained untreated or were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNF1 , CNFY or the N - or C - terminally deleted toxin variants at 37 ° C for 4 h . The cells were thoroughly washed , pelleted , lysed and the toxin variants bound to the cells were identified by western blotting using an anti - Flag antibody .

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Figure 2HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNF1, CNFY or the N- or C-terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody.


Figure 3Left upper and right panel : HEp - 2 cells re mained untreated or were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY or the N - or C - terminally deleted toxin variants for 4 h . Cells were lysed and the de amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels ; left lower panel : HEp - 2 cells were lysed and the cell extracts were incubated with full - length CNFY or the N - ter minally deleted toxin variants for 4 h . The de amidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti - RhoA antibodies . HEp - 2 cells were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY or the N - or C - terminally deleted toxin variants for 24 h . The cell nuclei were strained with DAPI ( blue ) and the actin cytoskeleton was stained using FITC - phalloidin ( green ) . The formation of large , multinuclear cells was ob served by fluorescence micro scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY - treated cells . The white scale bar is 40 µm . Cells incubated with extracts of YP147 ( ΔcnfY ) harboring the empty expression vector were used as negative controls .

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Figure 3Left upper and right panel: HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; left lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNFY or the N-terminally deleted toxin variants for 4 h. The deamidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti-RhoA antibodies.HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.


Figure 4HEp - 2 cells were incubated with 20 µg / ml of whole cell extract of Y . pseudotuberculosis expressing full - length CNFY , N - or C - terminal deletion variants fused to GFP ( green ) for 90 or 180 min . Cells were fixed and processed for fluorescence microscopy . The red fluorescent signal represents late endosomes ( CellLight Late Endosomes - RFP ( Rab7a ) ) . Nuclei were stained with DAPI ( blue ) . A merged image of the different channels is shown , and smaller images are magnified views of boxed areas . White scale bar is 10 µm .

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Figure 4HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY, N- or C-terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.


Figure 6Nitrocefin ( 2 mM ) was added to the supernatant from 25 ° C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β - lactamase activity by measuring changes in absorbance at 390 nm ( yellow ) and 486 nm ( red ) . The data represent the mean ± SD of three independent experiments , carried out in triplicates . The viability of Y . pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter - Glo Microbial Cell Viability Assay kit ( Promega ) . The data represent the mean ± SD of three independent experiments , carried out in triplicates .

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Figure 6Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.The viability of Y. pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega). The data represent the mean ± SD of three independent experiments, carried out in triplicates.


Figure 7Comparative analysis of RhoA activation in HEp - 2 cell lysate by CNFY and the recombinant D4 - 5 protein . Purified CNFY or the D4 - 5 fragment ( 1 µM ) was added to extracts of HEp - 2 cells and incubated for 10 , 20 , 30 or 60 min . De amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti - RhoA antibodies . Comparative analysis of recombinant RhoA deamidation by CNFY and the D4 - 5 fragment . Recombinant RhoA was incubated with purified CNFY or the D4 - 5 fragment and samples were separated by SDS - PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry . Error bars represent standard deviations of triplicate measurements .

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

Figure 7Comparative analysis of RhoA activation in HEp-2 cell lysate by CNFY and the recombinant D4-5 protein. Purified CNFY or the D4-5 fragment (1 µM) was added to extracts of HEp-2 cells and incubated for 10, 20, 30 or 60 min. Deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti-RhoA antibodies.Comparative analysis of recombinant RhoA deamidation by CNFY and the D4-5 fragment. Recombinant RhoA was incubated with purified CNFY or the D4-5 fragment and samples were separated by SDS-PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.


Figure 3The number of neoblasts did not change significantly following inhibition of the MTC - gene kiaa1429 . Shown is FISH of smedwi - 1 ( A ) in control and kiaa1429 ( RNAi ) animals ( left ) . smedwi - 1 + cells were counted following FISH and showed no significant difference in expression ( right ; bar represents average normalized cell count ; Materials and Methods ) . Scale = 100 µm . The number of neoblasts did not change significantly following inhibition of the MTC - gene kiaa1429 . Shown is FISH of h2b ( B ) in control and kiaa1429 ( RNAi ) animals ( left ) . h2b + cells were counted following FISH and showed no significant difference in expression ( right ; bar represents average normalized cell count ; Materials and Methods ) . Scale = 100 µm . ( E ) Representative FACS plots of cells isolated from control ( top ) and kiaa1429 ( RNAi ) animals ( bottom ) show an increase in cell abundance in the X2 ( Fincher et al , 2018 ) gate ( green ; Materials and Methods ) . ( F ) Quantification of the cells in each FACS gate in control and kiaa1429 ( RNAi ) animals showed a six - fold increase in the abundance of cells in the X2 gate ( Student ' s t - test * * * - p < 0 . 005 . FACS experiments were performed in biological triplicates . Boxes represent the IQR , whiskers represent the 1 . 5 x IQR , and central band represents the

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

Figure 3The number of neoblasts did not change significantly following inhibition of the MTC-gene kiaa1429. Shown is FISH of smedwi-1 (A) in control and kiaa1429 (RNAi) animals (left). smedwi-1+ cells were counted following FISH and showed no significant difference in expression (right; bar represents average normalized cell count; Materials and Methods). Scale = 100 µm.The number of neoblasts did not change significantly following inhibition of the MTC-gene kiaa1429. Shown is FISH of h2b (B) in control and kiaa1429 (RNAi) animals (left). h2b+ cells were counted following FISH and showed no significant difference in expression (right; bar represents average normalized cell count; Materials and Methods). Scale = 100 µm.(E) Representative FACS plots of cells isolated from control (top) and kiaa1429 (RNAi) animals (bottom) show an increase in cell abundance in the X2 (Fincher et al, 2018) gate (green; Materials and Methods). (F) Quantification of the cells in each FACS gate in control and kiaa1429 (RNAi) animals showed a six-fold increase in the abundance of cells in the X2 gate (Student's t-test *** - p < 0.005. FACS experiments were performed in biological triplicates. Boxes represent the IQR, whiskers represent the 1.5 x IQR, and central band represents the med


figf1 ( a ) MCF7 cells stably expressing Myc - TEV - Flag - Beclin 1 ( MEF - Beclin 1 ) and parental controls were lysed and subjected to MEF - tag - based purification . Proteins bound to MEF - Beclin 1 were isolated and detected by SDS - PAGE and silver staining . Seven bands specific for MEF - Beclin 1 are numbered . ( b ) Six bands were identified by nanoflow LC - MS / MS and MASCOT software . ( c ) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies . ( d ) Differential subcellular fractionation . Post - nuclear supernatant of A549 cell lysates were spun at 20 , 000g for 10 min and the supernatant was further spun at 100 , 000g for 60 min . Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies , including anti - Sec61β ( ER ) , cathepsin D ( lysosome ) , EEA1 ( early endosome ) , transferrin receptor ( plasma membrane and recycling endosome ) and GAPDH ( cytosol ) . ( e ) Gel filtration analysis . The supernatant fraction of A549 cell ( or Atg14L knockdown ) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody ( see Supplementary Information , Fig . S2e ) . Relative amounts of each fraction determined by densitometry were plotted . Vo , void fraction . The elution pattern of each protein was reproduced in several experiments . Full scans of the gel and blots in a , c and d are available in Supplementary Information , Fig . S6 .

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

figf1(a) MCF7 cells stably expressing Myc-TEV-Flag-Beclin 1 (MEF-Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification. Proteins bound to MEF-Beclin 1 were isolated and detected by SDS-PAGE and silver staining. Seven bands specific for MEF-Beclin 1 are numbered. (b) Six bands were identified by nanoflow LC-MS/MS and MASCOT software.(c) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies.(d) Differential subcellular fractionation. Post-nuclear supernatant of A549 cell lysates were spun at 20,000g for 10 min and the supernatant was further spun at 100,000g for 60 min. Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies, including anti-Sec61β (ER), cathepsin D (lysosome), EEA1 (early endosome), transferrin receptor (plasma membrane and recycling endosome) and GAPDH (cytosol).(e) Gel filtration analysis. The supernatant fraction of A549 cell (or Atg14L knockdown) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody (see Supplementary Information, Fig. S2e). Relative amounts of each fraction determined by densitometry were plotted. Vo, void fraction. The elution pattern of each protein was reproduced in several experiments. Full scans of the gel and blots in a, c and d are available in Supplementary Information, Fig. S6.


figf2 ( a ) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L , Rubicon , UVRAG or a control ( luciferase ) . Cell lysates were subjected to immunoblotting with the antibodies indicated . The asterisk in the Rubicon blot represents a non - specific cross - reacting band . ( b ) Quantification of GFP - positive dots per cell . Images were taken from cells shown in c and the number of GFP - positive dots was determined . At least 140 cells were counted per experiment and the values are mean ± s . d . of three independent experiments . ( c ) A monoclonal A549 cell line stably expressing GFP - LC3 was infected with adenovirus harbouring shRNA targeting Atg14L , Rubicon or control ( luciferase ) . The cells were cultured under nutrient - rich or starvation conditions . GFP was observed by confocal laser microscopy . Scale bar , 10 μm . The full scan of the blot in a is available in Supplementary Information , Fig . S6 .

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figf2(a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase). Cell lysates were subjected to immunoblotting with the antibodies indicated. The asterisk in the Rubicon blot represents a non-specific cross-reacting band.(b) Quantification of GFP-positive dots per cell. Images were taken from cells shown in c and the number of GFP-positive dots was determined. At least 140 cells were counted per experiment and the values are mean ± s.d. of three independent experiments. (c) A monoclonal A549 cell line stably expressing GFP-LC3 was infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon or control (luciferase). The cells were cultured under nutrient-rich or starvation conditions. GFP was observed by confocal laser microscopy. Scale bar, 10 μm. The full scan of the blot in a is available in Supplementary Information, Fig. S6.


figf3 ( a ) Southern blot analysis of genomic DNA isolated from wild - type ( WT , mAtg14L + / + ) , mAtg14L + / - and knockout ( KO , mAtg14L - / - ) ES cells after digestion with PstI . Details of the probe are shown in Supplementary Information , Fig . S3 . ( c ) The effect of Atg14L knockout on p62 turnover . WT , mAtg14L − / − , mAtg14L − / − + Atg14L , and mAtg5 − / − ES cells were cultured in nutrient - rich ( N ) or starvation medium ( S ) for 3 h . Cell lysates were subjected to western blotting with the antibodies indicated . ( d ) Degradation of long - lived proteins is reduced in Atg14L knockout cells . WT , mAtg14L − / − , and mAtg5 − / − ES cells were cultured in N or S medium or S medium with wortmannin ( W , 100 nM ) for 3 h and degradation of long - lived proteins was measured . The data are the mean ± s . d . of three independent experiments . ( e ) The effect of Atg14L knockout on LC3 turnover . WT , mAtg14L − / − and mAtg5 − / − ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d ( 50 μg ml − 1 ) and pepstatin A ( 50 μg ml − 1 ) . Cell lysates were subjected to western blotting with the antibodies indicated . Full scans of the blots in b , c and e are available in Supplementary Information , Fig . S6 .

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figf3(a) Southern blot analysis of genomic DNA isolated from wild-type (WT, mAtg14L+/+) , mAtg14L+/- and knockout (KO, mAtg14L-/-) ES cells after digestion with PstI. Details of the probe are shown in Supplementary Information, Fig. S3.(c) The effect of Atg14L knockout on p62 turnover. WT, mAtg14L−/−, mAtg14L−/− + Atg14L, and mAtg5−/− ES cells were cultured in nutrient-rich (N) or starvation medium (S) for 3 h. Cell lysates were subjected to western blotting with the antibodies indicated.(d) Degradation of long-lived proteins is reduced in Atg14L knockout cells. WT, mAtg14L−/−, and mAtg5−/− ES cells were cultured in N or S medium or S medium with wortmannin (W, 100 nM) for 3 h and degradation of long-lived proteins was measured. The data are the mean ± s.d. of three independent experiments.(e) The effect of Atg14L knockout on LC3 turnover. WT, mAtg14L−/− and mAtg5−/− ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1). Cell lysates were subjected to western blotting with the antibodies indicated. Full scans of the blots in b, c and e are available in Supplementary Information, Fig. S6.


figf4 ( a ) The effect of Atg14L knockout ( mAtg14 − / − ) on Atg16L dot formation . Wild - type ( WT ) and mAtg14 − / − ES cells were cultured in nutrient - rich or starvation medium for 3 h . Cells were fixed and subjected to immunofluorescence with an anti - Atg16L antibody . Images of Atg16L staining ( grey - scale ) and Atg16L merged with Hoechst staining ( colour ) are shown . ( c ) Quantification of the number of Atg16L dots in a . The data are the mean ± s . d . of three independent experiments . ( b ) The effect of Atg14L knockout on LC3 dot formation . WT and mAtg14L − / − ES cells were cultured in nutrient - rich or starvation medium for 3 h . Cells were fixed and subjected to immunofluorescence with anti - LC3 antibody . Images of LC3 staining ( grey - scale ) and LC3 merged with Hoechst staining ( colour ) are shown . Scale bars , 20 μm . ( d ) Quantification of the number of LC3 dots in b . The data are the mean ± s . d . of three independent experiments . For each of the experiments in c and d at least 13 cells were counted .

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

figf4(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown.(c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments.(b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.


figf5 ( a ) Immunofluorescence using an anti - Atg16L antibody . A549 cells were transfected with adenovirus expressing GFP - Atg14L and cultured in nutrient - rich or starvation medium for 4 h . Cells were fixed and stained with an anti - Atg16L antibody . ( b ) Immunofluorescence with an anti - LC3 antibody . A549 cells were transfected with adenovirus expressing GFP - Atg14L and cultured in starvation medium for 4 h . Cells were fixed and stained with an anti - LC3 antibody . Scale bar , 10 μm . ( c ) Colocalization efficiency in a and b . The percentage of colocalization between Atg14L and Atg16L or LC3 - positive dot is shown . For example , ( Atg14L × Atg16L ) / Atg16L means the ratio of colocalization of Atg14L and Atg16L puncta to all of the Atg16L - positive puncta . For colocalization between Atg14L and Atg16L the data are mean ± s . d . of 25 cells . For colocalization between Atg14L and LC3 the data are mean ± s . d . of 20 cells . d ) Immunofluorescence with an anti - calnexin ( ER ) antibody . A549 cells transfected with adenovirus - based GFP - Atg14L . After 48 h , the cells were fixed and immunostained with calnexin ( ER ) . Scale bar , 10 μm . ( e ) Immuno - EM analysis of GFP - Atg14L . A549 cells transfected with adenovirus - vector based GFP or GFP - Atg14L were subjected to immuno - EM analysis using an anti - GFP antibody . Scale bar , 500 nm . The arrows indicate the ER membrane and the arrowheads indicate the autophagosome membrane .

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figf5(a) Immunofluorescence using an anti-Atg16L antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in nutrient-rich or starvation medium for 4 h. Cells were fixed and stained with an anti-Atg16L antibody.(b) Immunofluorescence with an anti-LC3 antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in starvation medium for 4 h. Cells were fixed and stained with an anti-LC3 antibody. Scale bar, 10 μm.(c) Colocalization efficiency in a and b. The percentage of colocalization between Atg14L and Atg16L or LC3-positive dot is shown. For example, (Atg14L×Atg16L)/Atg16L means the ratio of colocalization of Atg14L and Atg16L puncta to all of the Atg16L-positive puncta. For colocalization between Atg14L and Atg16L the data are mean ± s.d. of 25 cells. For colocalization between Atg14L and LC3 the data are mean ± s.d. of 20 cells.d) Immunofluorescence with an anti-calnexin (ER) antibody. A549 cells transfected with adenovirus-based GFP-Atg14L. After 48 h, the cells were fixed and immunostained with calnexin (ER).Scale bar, 10 μm.(e) Immuno-EM analysis of GFP-Atg14L. A549 cells transfected with adenovirus-vector based GFP or GFP-Atg14L were subjected to immuno-EM analysis using an anti-GFP antibody. Scale bar, 500 nm. The arrows indicate the ER membrane and the arrowheads indicate the autophagosome membrane.


figf6 ( a ) Double knockdown of Rubicon and Atg14L . A549 cells stably expressing GFP - LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA ( luciferase ) . The image is available in Supplementary Information , Fig . S5a . The GFP - LC3 puncta were counted in nutrient - rich ( N ) or starvation ( S ) conditions . The data are mean ± s . d . of three independent experiments ; at least 23 cells were counted for each experiment . ( b ) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control ( luciferase ) . The cells were cultured in N or S medium in the presence or absence of E64d ( 50 μg ml − 1 ) and pepstatin A ( 50 μg ml − 1 ) for 1 - 4 h . Cell lysates were subjected to western blotting with the antibodies indicated . ( c ) Quantification of Atg16L puncta in Rubicon knockdown cells . The images are available in Supplementary Information , Fig . S5c . The data are mean ± s . d . , n = 60 ( 60 cells , sample of Medium : N , shRNA : Control ) , n = 68 ( 68 cells , sample of Medium : S , shRNA : Control ) , n = 78 ( 78 cells , sample of Medium : N , shRNA : Rubicon ) , n = 83 ( 83 cells sample of Medium : S , shRNA : Rubicon ) . d ) Bulk degradation activity of Rubicon knockdown cells . The bulk degradation activity in control and Rubicon knockdown cells in nutrient - rich medium was measured . Wortmannin ( W , 100 nM ) was added as a negative control . The data are mean ± s . d . of three independent experiments . ( e ) A549 cells infected with adenovirus harbouring shRNA for Rubicon and control ( luciferase ) were cultured in N or S medium for 4 h and cell lysates were subjected to immunoblotting with the antibodies indicated . Full scans of the blots in b and e are available in Supplementary Information , Fig . S6 .

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figf6(a) Double knockdown of Rubicon and Atg14L. A549 cells stably expressing GFP-LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA (luciferase). The image is available in Supplementary Information, Fig. S5a. The GFP-LC3 puncta were counted in nutrient-rich (N) or starvation (S) conditions. The data are mean ± s.d. of three independent experiments; at least 23 cells were counted for each experiment.(b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1-4 h. Cell lysates were subjected to western blotting with the antibodies indicated.(c) Quantification of Atg16L puncta in Rubicon knockdown cells. The images are available in Supplementary Information, Fig. S5c. The data are mean ± s.d., n = 60 (60 cells, sample of Medium: N, shRNA: Control), n = 68 (68 cells, sample of Medium: S, shRNA: Control), n = 78 (78 cells, sample of Medium: N, shRNA: Rubicon), n = 83 (83 cells sample of Medium: S, shRNA: Rubicon).d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments.(e) A549 cells infected with adenovirus harbouring shRNA for Rubicon and control (luciferase) were cultured in N or S medium for 4 h and cell lysates were subjected to immunoblotting with the antibodies indicated. Full scans of the blots in b and e are available in Supplementary Information, Fig. S6.


figf7 ( a ) A549 cells transfected with a GFP - Rubicon expression plasmid and were subjected to immunofluorescence with various organelle marker antibodies . Scale bar , 10 μm . ( b ) A549 cells stably expressing GFP - LC3 were subjected to Rubicon knockdown ( KD ) and then transfected with mStrawberry , mStrawberry - Rubicon or mStrawberry - Rubicon mutant lacking the Beclin 1 binding region ( Δ393849 ) . Cells with strong expression of mStrawberry - Rubicon showed an overexpression - dependent dominant - negative phenotype ; therefore , we selected cells with modest expression . The number of GFP - LC3 puncta was counted and mean ± s . d . are shown . mStrawberry , n = 12 ( 12 cells ) ; mStrawberry - Mm - Rubicon , n = 14 ( 14 cells ) ; mStrawberry - Mm - RubiconΔ393 - 849 , n = 13 ( 13 cells ) .

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figf7(a) A549 cells transfected with a GFP-Rubicon expression plasmid and were subjected to immunofluorescence with various organelle marker antibodies. Scale bar, 10 μm.(b) A549 cells stably expressing GFP-LC3 were subjected to Rubicon knockdown (KD) and then transfected with mStrawberry, mStrawberry-Rubicon or mStrawberry-Rubicon mutant lacking the Beclin 1 binding region (Δ393849). Cells with strong expression of mStrawberry-Rubicon showed an overexpression-dependent dominant-negative phenotype; therefore, we selected cells with modest expression. The number of GFP-LC3 puncta was counted and mean ± s.d. are shown. mStrawberry, n = 12 (12 cells); mStrawberry-Mm-Rubicon, n = 14 (14 cells); mStrawberry-Mm-RubiconΔ393-849, n = 13 (13 cells).


figf8 ( a ) The maturation of autophagosomes is enhanced in Rubicon knockdown cells . A549 cells stably expressing GFP - LC3 , with Rubicon or control shRNA , were incubated in nutrient - rich medium with or without the protease inhibitors ( PI ) E64d ( 50 μg ml − 1 ) and pepstatin A ( 50 μg ml − 1 ) treatment for 4 h . The cells were stained with an anti - Lamp1 antibody ( upper panels ) and colocalization efficiency between GFP - LC3 and Lamp1 was counted ( lower panel ) . The data are mean ± s . d . of 30 cells . ( b ) . Electron microscopy analysis of Rubicon knockdown cells . Control ( luciferase ) or Rubicon shRNA A549 cells grown in nutrient - rich medium was subjected to electron microscopy analysis ( left panels ) . The arrows indicate autophagosomes and the arrowheads indicate autolysosomes . Scale bar , 1 μm . Autophagosomes and autolysosomes were counted in 21 cells , and the mean ± s . d . are shown ( right panel ) . ( c ) EGFR degradation in Rubicon knockdown cells . Control or Rubicon shRNA A549 cells were treated with EGF ( 200 ng ml − 1 ) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated ( left panel ) . The band intensity was measured in three independent experiments and the mean ± s . d . are shown ( right panel ) . ( d ) The effect of Rubicon overexpression in the turnover of EGF receptor . A549 cells were transfected with advenovirus - based GFP or GFP - Rubicon . After 24 h , EGF was added to cells expressing GFP - Rubicon or control GFP . At the times indicated , the cells were lysed and subjected to western blotting with the indicated antibodies . ( d ) The effect of Rubicon overexpression in the turnover of EGF receptor . A549 cells were transfected with advenovirus - based GFP or GFP - Rubicon . After 24 h , EGF was added to cells expressing GFP - Rubicon or control GFP . At the times indicated , the cells were lysed and subjected to western blotting with the indicated antibodies .

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figf8(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP-LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP-LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells.(b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel).(c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel).(d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.(d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.


Figure 1Kaplan - Meier curves for overall survival ( OS ) in lung adenocarcinoma TCGA _ LUAD ( RASSF1 mRNA high / low cutoff FKPM 5 . 85 ) and squamous cell carcinoma patients TCGA _ LUSC ( RASSF1 mRNA high / low cutoff FKPM 6 . 52 ) . Significance derived from log - rank test . Western blot with indicated antibodies of isogenic H1299 cells stably transfected with either empty vector pcDNA3 ( H1299control ) or RASSF1A ( H1299RASSF1A ) . Bottom : cell proliferation resazurin assay . ( n = 2 ) . Error bars represent mean ± S . E . M . Size of lung primary tumours measured at day 30 after lung orthotopic injection either with H1299control ( n = 14 mice per group ; experimental groups of 6 and 8 different shading within the groups represents two independent experiments ) or with H1299RASSF1A cells stably re - expressing RASSF1A , ( n = 12 mice per group ; experimental groups of 6 and 6 ) . The size of primary tumours was measured by MRI software ITK - SNAP . Statistical significance via 2 - tailed Student ' s t - test . Error bars represent mean ± S . E . M . Visual assessment quantification of metastatic events on the ipsilateral ( left ) and or contralateral ( right ) lungs , generated by H1299control or H1299RASSF1A at day 30 . Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A . Statistical significance via 2 - tailed Student ' s t - test . Error bars represent mean ± S . E . M .

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Figure 1Kaplan-Meier curves for overall survival (OS) in lung adenocarcinoma TCGA_LUAD (RASSF1 mRNA high/low cutoff FKPM 5.85) and squamous cell carcinoma patients TCGA_LUSC (RASSF1 mRNA high/low cutoff FKPM 6.52). Significance derived from log-rank test.Western blot with indicated antibodies of isogenic H1299 cells stably transfected with either empty vector pcDNA3 (H1299control) or RASSF1A (H1299RASSF1A). Bottom: cell proliferation resazurin assay. (n=2). Error bars represent mean ± S.E.M.Size of lung primary tumours measured at day 30 after lung orthotopic injection either with H1299control (n=14 mice per group; experimental groups of 6 and 8 different shading within the groups represents two independent experiments) or with H1299RASSF1A cells stably re-expressing RASSF1A, (n=12 mice per group; experimental groups of 6 and 6). The size of primary tumours was measured by MRI software ITK-SNAP. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.Visual assessment quantification of metastatic events on the ipsilateral (left) and or contralateral (right) lungs, generated by H1299control or H1299RASSF1A at day 30. Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.


Figure 2Mass - spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results . Only proteins identified with two or more peptides were taken into consideration . The resulting list of proteins was restricted to ECM proteins ( n = 3 ) . Kaplan Meier plots depicting prognosis in TCGA _ LUAD ( lung adenocarcinoma ) with high and low mRNA expression of ECM proteins revealed by proteomics . Significance derived from log - rank test . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M . Representative images of H & E and IHC staining for P4HA2 , and quantification ( bars ) of two independent regions of n = 5 H1299control and n = 3 H1299RASSF1A primary lung tumours . Scale bars 100µm . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M . Hydroxyproline assay ( Cell biolabs Inc . ) for total hydroxyproline activity of cells . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M . Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells . Western blot indicating levels of Collagen I in H1299control cells in the presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination . Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination . H1299RASSF1A cell line was used as negative control for comparison . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M . Immunofluorescence staining for Collagen I and P4HA2 and quantification ( right graph ) in HOP92 cells after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M . Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A . Immunofluorescence staining for Collagen I and P4HA2 and quantification ( right graph ) in HeLa cells after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated all statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 experiments and error bars represent the mean ± S . E . M .

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Figure 2Mass-spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n=3).Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells.Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.