Figure 6A By immunoblot analysis , the levels of Rabaptin5 and as a loading control of tubulin ( Tub ) were assessed in wild - type HEK293A cells , HEK + Rbpt5 cells stably overexpressing Rabaptin5 , and Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) . B The same stable HEK293A - derived cell lines were treated without ( - CQ ) or with 60 µM chloroquine for 150 min ( + CQ ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B . Scale bar , 10 µm . CD , E The HEK293A - derived cell lines were treated with 60 µM chloroquine for 30min and non - lipidated and lipidated LC3B ( I and II , resp . ) were assayed by immunoblot analysis ( D ) . The increase of the LC3B - II fraction of total LC3B upon chloroquine treatment was quantified ( mean ± S . D . of three independent experiments ; F , G HEK + Rbpt5 cells and Rabaptin5 - knockout cells stably re - expressing wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were transfected with mCherry - ATG16L1 , treated with 60 µM chloroquine ( CQ ) for 0 , 15 and 30 min , and analyzed by immunofluorescence microscopy for Rabaptin5 , mCherry - ATG16L1 , and WIPI2 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for mCherry - ATG16L1 ( F ) or WIPI2 ( G ) and M2 showing the inverse ( mean ± S . D . of three independent experimentsH Wild - type HEK293A cells , HEK + Rbpt5 cells , Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were stained with lysotracker and with DAPI for nuclei . Scale bar , 10 µm . I The number of lysosomes ( lysotracker - positive structures ) per cell was quantified from cells as in panel C ( mean ± S . D . of three independent experiments ) . Images for at least 15 cells per sample were quantified . The value for HEK + Rbpt5 cells is an underestimate , because the density of puncta makes them difficult to distinguish as separate structures .

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

Figure 6A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm. CD, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments;F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experimentsH Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm. I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures.


Figure 7A HeLa cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h . The cells were infected with Salmonella by centrifugation at 500 × g for 5min at 37 ° C and incubation for 10 min at 37 ° C , washed three times , and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0 , 1 , 3 , or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points , shown as a percentage of internalized cells after infection ( mean ± S . D . of three independent experiments ) . The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 ) . On the right , the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP , washed , and immediately fixed for fluorescence microscopy and stained with anti - transferrin receptor and anti - LC3B as cellular markers . Z - stacks for > 5 ' 000cells / sample were acquired and analyzed in Fiji to determine the fraction of infected cells ( mean ± S . D . of three independent experiments ) . The average number of bacteria per infected cell was identical ( 2 . 16 , 2 . 11 , and 2 . 12 bacteria per cell transfected with siCtr , siRbpt5 , and siFIP200 , respectively ) . B Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + wt , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella and treated and analyzed as in panels A ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * * * P < 0 . 0001 ) . C Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella expressing GFP as in panel B , incubated in fresh culture medium containing gentamicin for 0 , 5 , 15 , 30 , and 60 min , fixed with methanol and immunostained for transferrin receptor ( TfR ) as a marker of early endosomes and for LC3B as a marker of autophagy . Salmonella were classified according to their association with a TfR - and / or LC3B - positive compartment - as illustrated on the top left ( scale bar , 2 µm ) - during the first hour after infection . In the absence of Rabaptin5 , LC3 - positive SCVs with early endosomal characteristics ( containing TfR ) were strongly reduced . ( Mean ± S . D . of three independent experiments , analyzing > 50 bacteria for each time point . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).B Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ****P<0.0001).C Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)


Figure 2B . Volcano plot showing the magnitude of differential gene up - regulation , as measured by the respective log 2 - fold change in expression level , at day ( positive ) or night ( negative ) versus statistical significance , as quantified by a negative binomial exact test ( red dots , dashed line p < 0 . 05 adjusted for multiple testing using Benjamin - Hochberg ) . Known circadian - regulated genes are labelled .

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Figure 2B. Volcano plot showing the magnitude of differential gene up-regulation, as measured by the respective log 2-fold change in expression level, at day (positive) or night (negative) versus statistical significance, as quantified by a negative binomial exact test (red dots, dashed line p<0.05 adjusted for multiple testing using Benjamin-Hochberg). Known circadian-regulated genes are labelled.


Figure 3B . Violin plots depicting the distribution of the quantified average cellular expression of Prok2 , ProkR2 , Vip , Vipr2 , Avp , Avpr1a , Grp and Grpr in each sub - cluster in CT7 . 5 ( left ) and CT15 . 5 ( right ) datasets .

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Figure 3B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets.


Figure 5 C . The period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) . D . Similarly , the amplitude of Cry1 - luc bioluminescence rhythms from Prok2 + SCN cells and ProkR2 + SCN cells , does not differ between treatments ( p = 0 . 67 ) . H . Left : confocal images of representative SCN slices dual - transfected with pProk2 . Cre . T2A . mCherry and Syn . GCaMP6f ( top ) , or pProkR2 . Cre . T2A . Venus and Syn . jRCaMP1a ( below ) ( scale bar = 500µm ) . Right , above : representative detrended plots of signal from GCaMP6f ( green ) traces and pCry1 - DIO - Luc , emitted from Prok2 + SCN cells ( red ) . Right , below : representative detrended plots of jRCaMP1a ( purple ) and pCry1 - DIO - Luc emitted from ProkR2 + cells ( light green ) . Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN .

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

Figure 5 C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively). D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).  H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.


Figure 7 B . Comparison of the Per2 : : Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle ( applied CT23 - 20 dark grey n = 12 SCN slices , CT - 20 - 23 light grey n = 6 SCN slices ) or Prok2RA ( applied CT23 - 20 purple n = 13 SCN slices , CT20 - 23 pink n = 11 SCN slices ) treatment ( interaction , p < 0 . 0001 ) . A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20 - 23 ( * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).


Figure 8 H . Period lengthening occurred at the same rate when Prok2 . Cre or ProkR2 . Cre was the first AAV to be applied ( p = 0 . 1 ; Prok2 . Cre - baseline vs . 1 AAV , * p = 0 . 01 ; baseline vs . both AAVs , * p = 0 . 04 ; ProkR2 . Cre - baseline vs . 1 AAV , * p = 0 . 03 ; baseline vs . both AAVs , * * p = 0 . 01 .

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Figure 8  H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.


Figure 9 A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + cells ( red ) and further complementation in ProkR2 + cells ( yellow , rhythmic ) ( cohort n = 8 ) . B . as in A but order of complementation reversed ( green = Cry1 complementation in ProkR2 + cells ) ( cohort n = 8 ) .

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Figure 9 A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8). B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).


Figure 1Schematic representation of the upstream region of myogenin and amplified regions by RT - PCR ( top ) . RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes ( bottom ) . The presence or absence of reverse transcriptase ( RT ) is shown by ( + ) or ( - ) , respectively . The templates ( cDNA or genomic DNA ) are indicated by C or G , respectively . The primers used for RT - PCR ( top ) . Strand - specific RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA ( middle and bottom ) and poly ( A ) + RNA ( bottom ) . Relative expression of indicated RNAs in differentiating C2C12 cells . The presence or absence of RT is shown by ( + ) or ( - ) , respectively . PCR products were verified by sequencing . n = 3 , mean ± SD . The nuclear / cytoplasmic fractionation is confirmed by the expression of H2B and tubulin , respectively . Chromatin pellet extract ( CPE ) / soluble nuclear extract ( SNE ) ratio of SRA1 , Malat1 , and Myoparr in C2C12 myotubes on a log scale . n = 3 , mean ± SD .

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

Figure 1Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.


Figure 2Quantitative RT - PCR for myogenin ( A ) and Myoparr ( B ) during myogenesis of C2C12 cells , primary mouse myoblasts ( growth medium , GM ) , and mouse embryonic skeletal muscle ( S . K . ) . The x - axis shows days after differentiation induction or embryonic days . n = 3 , mean ± SD . qRT - PCR showing increased Myoparr expression by MyoD in C3H10T1 / 2 fibroblasts . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) . Treatment of recombinant TGF - β for 24 h decreased Myoparr expression in differentiating C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) . Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) . Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells by TGF - β treatment . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed 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 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 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 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 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 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

Figure 2Quantitative RT-PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x-axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.qRT-PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Welch's t-test).Treatment of recombinant TGF-β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells by TGF-β treatment. n = 3, mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


Figure 3C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium . After additional 24 - h incubation in growth medium , cells were transferred to differentiation medium . The levels of Myoparr and myogenin expression were quantified by qRT - PCR 24 h after differentiation induction . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant . Statistical analyses were performed using unpaired two - tailed Student ' s t - test . In cases of unequal variances , unpaired two - tailed Welch ' s t - test was used . Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD . Tubulin expression served as an internal control . The expression levels of myogenin and Myoparr evaluated by qRT - PCR in myogenin - depleted C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) . ChIP - qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) . Retrieval rate of Myoparr using Myoparr - ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values . GAPDH was amplified as a negative control . n = 3 , mean ± SD . ND , not detected . ChIRP - qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter . The myogenin 3 ´ UTR and GAPDH promoter were amplified as negative controls . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) . ND , not detected . Immunocytochemistry for the detection of myogenin at 24 h ( G ) or MHC at 72 h ( H ) in Myoparr - depleted C2C12 cells after differentiation induction . Nuclei were counterstained with DAPI . Bar , 100 μm . The percentage of myogenin - positive cells or fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) . Genes significantly differentially expressed in both Myoparr - and myogenin - depleted cells show correlated expression ( R = 0 . 63 , log 2 ratio scale ) .

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 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 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 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 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 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

Figure 3C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test).ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected.Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale).


Figure 4Following immunoprecipitation , the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17 - specific antibody . Two different Ddx17 isoforms ( p72 and p82 ) were observed . Determination of the Ddx17 - binding region of Myoparr by RNA pull - down analyses . Schematic diagram of full - length or truncated Myoparr used for RNA pull - down ( top ) . In vitro transcribed / translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody ( bottom ) . Relative luciferase activities of indicated constructs in differentiating C2C12 cells . Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA ( Myoparr KD ) .

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Figure 4Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom).Relative luciferase activities of indicated constructs in differentiating C2C12 cells.Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).


Figure 5Significantly decreased MHC expression in Ddx17 - depleted C2C12 cells is shown by immunocytochemistry . Nuclei were counterstained with DAPI . Bar , 100 μm . Fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Immunocytochemistry for myogenin in Ddx17 - depleted C2C12 cells . Bar , 100 μm . Myogenin - positive cells are shown as percent of the control . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Relative luciferase activities of the - 1650 - Luc by the combination of MyoD and Ddx17 or Ddx17 mutant ( K142R ) . Bars indicate the average of two independent experiments , and open circles represent the values of each experiment . Ddx17 or Ddx17 mutant ( K142R ) was pulled down by full - length Myoparr and then detected by western blot . Reduced interaction between endogenous Ddx17 and PCAF in Myoparr - depleted C2C12 cells . After Myoparr KD , the cell lysates were subjected to immunoprecipitation ( IP ) with a Ddx17 - specific antibody 36 h after differentiation induction ( right panel ) . Each lysate was loaded as an input ( left panel ) . Relative quantification of ( E ) is shown as IP / input ratio . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) . Increased interaction between Ddx17 and PCAF by Myoparr . PCAF protein was pulled down by 3 × Flag - Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot . ChIP - qPCR detection of Ddx17 ( H ) and PCAF ( I ) occupancies at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).Relative luciferase activities of the -1650-Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.Ddx17 or Ddx17 mutant (K142R) was pulled down by full-length Myoparr and then detected by western blot.Reduced interaction between endogenous Ddx17 and PCAF in Myoparr-depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17-specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel). Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *p < 0.05. n.s., not significant (unpaired two-tailed Student's t-test).Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3×Flag-Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot.ChIP-qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Figure 6Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr - or Ddx17 - depleted cells ( log 2 ratio scale ) . Genes significantly differentially expressed in both Myoparr - and Ddx17 - depleted cells show correlated expression ( R = 0 . 94 , log 2 ratio scale ) . Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal . C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h . After differentiation induction , cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h . EdU - positive cells are shown as percent of the control . Nuclei were counterstained with Hoechst 33342 . n = 3 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test ) . Bar , 100 μm .

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

Figure 6Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr- or Ddx17-depleted cells (log 2 ratio scale).Genes significantly differentially expressed in both Myoparr- and Ddx17-depleted cells show correlated expression (R = 0.94, log 2 ratio scale).Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU-positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Bar, 100 μm.


Figure 7qRT - PCR for pri - miR - 133b , pri - miR - 206 , and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . ChIP - qPCR detection of Pol II occupancy at the indicated promoters in Myoparr - depleted C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . Myoparr KD decreased Pol II occupancy at the miR - 133b promoter with a marginal trend toward significance ( p = 0 . 052 ) . ChIP - qPCR detection of Ddx17 ( C ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . ChIP - qPCR detection of PCAF ( D ) occupancies at the indicated promoters in Myoparr - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . qRT - PCR showing decreased expression of miR - 133b and miR - 206 in both Myoparr - and Ddx17 - depleted C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . Western blots showing increased ERK1 / 2 activity ( pERK1 / 2 ) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD . Tubulin expression served as an internal control . Increased Pola1 expression detected by qRT - PCR . n = 3 , mean ± SD . * * p < 0 . 01 .

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Figure 7qRT-PCR for pri-miR-133b, pri-miR-206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *p < 0.05. **p < 0.01.ChIP-qPCR detection of Pol II occupancy at the indicated promoters in Myoparr-depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. Myoparr KD decreased Pol II occupancy at the miR-133b promoter with a marginal trend toward significance (p = 0.052).ChIP-qPCR detection of Ddx17 (C) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.ChIP-qPCR detection of PCAF (D) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.qRT-PCR showing decreased expression of miR-133b and miR-206 in both Myoparr- and Ddx17-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control.Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.


Figure 8Seven days after denervation , weights of innervated ( - ) and denervated ( + ) tibialis anterior ( TA ) muscles were measured . n = 4 , mean ± SEM . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . Expression of myogenin in innervated and denervated TA muscles detected by qRT - PCR 7 d after denervation . n = 4 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Welch ' s t - test ) . qRT - PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation . n = 3 , mean ± SD . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) . Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation . Expression of tubulin served as an internal control . Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA . Muscle weights were measured 7 d after denervation . n = 5 for each group , mean ± SEM . * * * p < 0 . 001 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) . Representative immunostaining images for laminin ( red ) and EmGFP ( green ) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA , both containing EmGFP . Bar , 100 μm . Cross - sectional area ( CSA ) of electroporated EmGFP - positive myofibers ( 250 fibers per sample ) was analyzed 7 d after denervation . n = 5 for each group , mean ± SEM . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Figure 8Seven days after denervation, weights of innervated (-) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. *p < 0.05 (unpaired two-tailed Student's t-test).Expression of myogenin in innervated and denervated TA muscles detected by qRT-PCR 7 d after denervation. n = 4, mean ± SD. ***p < 0.001 (unpaired two-tailed Welch's t-test). qRT-PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation. n = 3, mean ± SD. ***p < 0.001 (unpaired two-tailed Student's t-test).Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation. Expression of tubulin served as an internal control.Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 d after denervation. n = 5 for each group, mean ± SEM. ***p < 0.001. n.s., not significant (unpaired two-tailed Student's t-test).Representative immunostaining images for laminin (red) and EmGFP (green) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA, both containing EmGFP. Bar, 100 μm.Cross-sectional area (CSA) of electroporated EmGFP-positive myofibers (250 fibers per sample) was analyzed 7 d after denervation. n = 5 for each group, mean ± SEM. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


Figure 3 ( A ) Immunohistochemistry of Bcl6 ( red ) , Cux1 ( green ) , and Satb2 ( blue ) in P2 Bcl11aF / F ; Emx1IRESCre and control neocortex . Nuclei are stained with Dapi ( white ) . ( B ) Relative quantification of Bcl6 + , Satb2 + , and Cux1 + cells in Bcl11aF / F ; Emx1IRESCre and control neocortex ( n = 4 ) . ( C ) Numbers of Cux1 + or Satb2 + cells that coexpress Bcl6 are reduced in Bcl11aF / F ; Emx1IRESCre compared to control neocortex ( n = 4 ) . Data information : All graphs represent the mean ± s . e . m . ; Student ' s t test ; * * * p < 0 . 001 . Scale bar , 50 µm .

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Figure 3(A) Immunohistochemistry of Bcl6 (red), Cux1 (green), and Satb2 (blue) in P2 Bcl11aF/F;Emx1IRESCre and control neocortex. Nuclei are stained with Dapi (white). (B) Relative quantification of Bcl6+, Satb2+, and Cux1+ cells in Bcl11aF/F;Emx1IRESCre and control neocortex (n = 4). (C) Numbers of Cux1+ or Satb2+ cells that coexpress Bcl6 are reduced in Bcl11aF/F;Emx1IRESCre compared to control neocortex (n = 4). Data information: All graphs represent the mean ± s.e.m.; Student's t test; *** p < 0.001. Scale bar, 50 µm.


Figure 4 ( C - E ) Immunohistochemistry of electroporated P2 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) and Bcl6 ( red , C ) , Cux1 ( red , D ) , or Satb2 ( red , E ) antibodies . Bcl6 expression is specifically downregulated Bcl11aF / F neocortex upon electroporation of CAG - CreGFP in comparison to CAG - CtlGFP control plasmid . Nuclei are stained with Dapi ( white ) . Data information : White arrowheads point at GFP + cells that also express Bcl6 , Cux1 , Satb2 indicate cells expressing only GFP + . Scale bars , 20 µm ( C - E ) , ( F ) Quantification of the percentage of electroporated cells expressing Bcl6 ( n = 3 ) , Satb2 ( n = 3 ) , and Cux1 ( n = 5 ) . Results are expressed as mean ± s . e . m . ; Student ' s t test ; * * * p < 0 . 001 . ( H ) Immunohistochemistry of electroporated P5 Bcl11a ∆ / F and Bcl11a + / F neurons in superficial cortical layers with GFP ( green ) and cleaved caspase 3 ( CC3 , magenta ) antibodies . Electroporation of Neurod - CreGFP plasmid together with CAG - LSL - Bcl6GFP into Bcl11aF / F neocortex reduces the number of CC3 + cells to control levels . ( I ) Quantification of the experiment shown in ( H ) ( n = 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc test ; * * * p < 0 . 001 . Data information : White arrowheads point at GFP + cells that express CC3 . Black arrowheads indicate cells expressing only GFP + . Scale bars , , 50 µm ( H ) .

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 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 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

Figure 4(C-E) Immunohistochemistry of electroporated P2 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and Bcl6 (red, C), Cux1 (red, D), or Satb2 (red, E) antibodies. Bcl6 expression is specifically downregulated Bcl11aF/F neocortex upon electroporation of CAG-CreGFP in comparison to CAG-CtlGFP control plasmid. Nuclei are stained with Dapi (white). Data information: White arrowheads point at GFP+ cells that also express Bcl6, Cux1, Satb2 indicate cells expressing only GFP+. Scale bars, 20 µm (C-E),(F) Quantification of the percentage of electroporated cells expressing Bcl6 (n = 3), Satb2 (n = 3), and Cux1 (n = 5). Results are expressed as mean ± s.e.m.; Student's t test; *** p < 0.001.(H) Immunohistochemistry of electroporated P5 Bcl11a∆/F and Bcl11a+/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of Neurod-CreGFP plasmid together with CAG-LSL-Bcl6GFP into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. (I) Quantification of the experiment shown in (H) (n = 4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; *** p < 0.001. Data information: White arrowheads point at GFP+ cells that express CC3. Black arrowheads indicate cells expressing only GFP+. Scale bars, , 50 µm (H).


Figure 5 ( A ) Immunohistochemistry of cleaved caspase 3 ( CC3 ) shows that the number of CC3 + cells ( marked by black arrowheads ) is increased in P5 Bcl6F / F ; NexCre compared to control neocortex . Insets are enlargements of the boxed areas in corresponding panels . Data information : Scale bars , 500 µm ( A ) ( C ) Quantification of the experiment shown in ( A ) ( n = 3 ) . Results are expressed as mean ± s . e . m . ; Student ' s t test ; * p < 0 . 05 . ( J ) Immunohistochemistry of electroporated P5 Bcl11aF / F neurons in superficial cortical layers with GFP ( green ) and cleaved caspase 3 ( CC3 , magenta ) antibodies . Electroporation of CAG - CreGFP plasmid together with Foxo1 - shRNAGFP # 4 into Bcl11aF / F neocortex reduces the number of CC3 + cells to control levels . White and black arrowheads indicate GFP + CC3 + and GFP + cells , respectively . ( K ) Quantification of the experiment shown in ( J ) ( n = 4 , CAG - CtlGFP / Ctl - shRNAGFP ; n = 3 , CAG - CreGFP / Ctl - shRNAGFP ; n = 5 , CAG - CreGFP / Foxo1 - shRNAGFP # 4 ) . Results are expressed as mean ± s . e . m . ; one - way ANOVA followed by Tukey ' s post hoc test ; * p < 0 . 05 . Data information : Scale bars , , 50 µm J ) .

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 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 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

Figure 5(A) Immunohistochemistry of cleaved caspase 3 (CC3) shows that the number of CC3+ cells (marked by black arrowheads) is increased in P5 Bcl6F/F;NexCre compared to control neocortex. Insets are enlargements of the boxed areas in corresponding panels. Data information: Scale bars, 500 µm (A)(C) Quantification of the experiment shown in (A) (n = 3). Results are expressed as mean ± s.e.m.; Student's t test; * p < 0.05.(J) Immunohistochemistry of electroporated P5 Bcl11aF/F neurons in superficial cortical layers with GFP (green) and cleaved caspase 3 (CC3, magenta) antibodies. Electroporation of CAG-CreGFP plasmid together with Foxo1-shRNAGFP#4 into Bcl11aF/F neocortex reduces the number of CC3+ cells to control levels. White and black arrowheads indicate GFP+ CC3+ and GFP+ cells, respectively. (K) Quantification of the experiment shown in (J) (n = 4, CAG-CtlGFP/Ctl-shRNAGFP; n = 3, CAG-CreGFP/Ctl-shRNAGFP; n = 5, CAG-CreGFP/Foxo1-shRNAGFP#4). Results are expressed as mean ± s.e.m.; one-way ANOVA followed by Tukey's post hoc test; * p < 0.05. Data information: Scale bars, , 50 µm J).


Figure 1The Cancer Genome Atlas ( TCGA RNAseq dataset ) was used on the GlioVis platform ( Bowman et al , 2007 ) to analyze the probability of survival ( log - rank p - value ) of 155 GBM patients , for each gene encoding for the mediators of the NF - κB pathway . Kaplan - Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level , using median cut - off , based on the TCGA RNAseq dataset . Box and whisker plot of MALT1 mRNA expression in low - grade glioma ( LGG , grades II and III ) or in GBM ( grade IV ) ( TCGA GBMLGG , RNAseq dataset ) ( D ) . Alternatively , MALT1 mRNA expression was plotted in non - tumor samples versus GBM samples ( TCGA RNAseq dataset ) ( E ) . Each dot represents one clinical sample . Fraction of surviving cells over time in GSC # 1 and GSC # 9 , transduced with control ( shc ) or bi - cistronic GFP plasmids using two different short hairpin RNA ( shMALT1 sequences , seq # 1 and # 2 ) . Data are plotted as the percentage of GFP - positive cells at the day of the analysis ( Dx ) , normalized to the starting point ( Day 4 post - infection , D4 ) . EdU incorporation ( green , 2 hours ) was visualized by confocal imagery in GSC # 1 or by FACS in GSC # 9 transfected with sic or siMALT1 . In GSC # 1 the percentage of EdU - positive cells was quantified . Nuclei ( DAPI ) are shown in blue . n > 240 cells per replicate . Scale bar : 10 μm . Data are presented as the mean + s . e . m . on 3 independent experiments . FACS analysis of propidium iodide ( PI ) staining in GSC # 1 and # 9 transfected with non - silencing duplexes ( sic ) or MALT1 siRNA duplexes ( siMALT1 ) and analyzed 72 hours later . Linear regression plot of in vitro limiting dilution assay ( LDA ) for control ( shc ) or shMALT1 seq # 1 and seq # 2 transduced GSC # 9 . Data are representative of n = 2 . Knockdown efficiency was verified at day 3 by western - blot using anti - MALT1 antibodies . GAPDH served as a loading control . Tumorspheres per field of view ( fov ) were manually counted in sic or siMALT1 transfected GSC # 1 , # 4 and # 9 . Data are presented as the mean + s . e . m . on 3 independent experiments .

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

Figure 1The Cancer Genome Atlas (TCGA RNAseq dataset) was used on the GlioVis platform (Bowman et al, 2007) to analyze the probability of survival (log-rank p-value) of 155 GBM patients, for each gene encoding for the mediators of the NF-κB pathway.Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level, using median cut-off, based on the TCGA RNAseq dataset.Box and whisker plot of MALT1 mRNA expression in low-grade glioma (LGG, grades II and III) or in GBM (grade IV) (TCGA GBMLGG, RNAseq dataset) (D).Alternatively, MALT1 mRNA expression was plotted in non-tumor samples versus GBM samples (TCGA RNAseq dataset) (E). Each dot represents one clinical sample.Fraction of surviving cells over time in GSC#1 and GSC#9, transduced with control (shc) or bi-cistronic GFP plasmids using two different short hairpin RNA (shMALT1 sequences, seq #1 and #2). Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4).EdU incorporation (green, 2 hours) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1 the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n> 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean + s.e.m. on 3 independent experiments.FACS analysis of propidium iodide (PI) staining in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 hours later.Linear regression plot of in vitro limiting dilution assay (LDA) for control (shc) or shMALT1 seq#1 and seq#2 transduced GSC#9. Data are representative of n=2. Knockdown efficiency was verified at day 3 by western-blot using anti-MALT1 antibodies. GAPDH served as a loading control.Tumorspheres per field of view (fov) were manually counted in sic or siMALT1 transfected GSC#1, #4 and #9. Data are presented as the mean + s.e.m. on 3 independent experiments.


Figure 2Linear regression plot of in vitro limiting dilution assay ( LDA ) for GSC # 9 treated with MALT1 inhibitor , mepazine ( MPZ , 20 μM , 14 days ) . DMSO vehicle was used as a control . Data are representative of n = 2 . Stem cell frequency was calculated from LDA in GSCs # 1 , # 4 , and # 12 treated with MPZ treatment ( 20 μM , 14 days ) . Data are presented as the mean + s . e . m . on 2 independent experiments . Tumorspheres per field of view ( fov ) were manually counted in GSCs # 1 , # 4 , # 9 , and # 12 in response to MPZ ( 20 μM ) and vehicle ( DMSO ) , and in GSC # 9 treated with Z - VRPR - FMK ( 75 µM ) and vehicle ( H2O ) for 4 days . Data are presented as the mean + s . e . m . on 4 independent experiments for MPZ and 3 independent experiments for Z - VRPR - FMK . The expression of the stemness markers SOX2 and NESTIN was evaluated by western - blot and immunofluorescence ( SOX2 in red NESTIN in green ) in MPZ ( + , 20 μM , 16 hours ) and vehicle ( - , DMSO , 16 hours ) treated GSC # 9 . GAPDH served as a loading control . Scale bar : 10 μm . Cell viability was measured using Cell TiterGlo luminescent assay in GSCs # 1 , # 4 , # 9 , and # 12 , human brain endothelial cells ( endo ) , human astrocytes ( astro ) , and human neuron - like cells ( neuron ) treated for 48 hours with DMSO or MPZ ( 20 μM ) . Data were normalized to their respective DMSO - treated controls and are presented as the mean + s . e . m of 3 independent experiments in triplicate . FACS analysis of EdU staining was performed on GSC # 1 treated overnight with MPZ ( 10 μM ) . Data are presented as the mean + s . e . m . on 3 independent experiments . FACS analysis of propidium iodide ( PI ) staining in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC # 1 # 4 , and # 9 ( DGCs ) treated for 48 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Data were normalized to their respective DMSO - treated controls and are presented as the mean + s . e . m of 3 independent experiments . Morphology of GSCs # 1 , # 4 , # 9 and DGCs # 1 , # 4 , # 9 was shown using brightfield images . Heatmap of cell viability of GSC # 9 using increasing doses ( 0 , 5 , 10 , 20 , 40 μM ) of phenothiazines : mepazine ( MPZ ) , fluphenazine ( FLU ) , cyamemazine ( CYAM ) , chlorpromazine ( CHLO ) , pipotiazine ( PIPO ) , alimemazine ( ALI ) , promethazine ( PRO ) , and doxylamine ( DOXY ) . Data were normalized to their respective DMSO - treated controls . Nude mice were implanted with GSC # 9 ( 106 cells ) in each flank and randomized cages were treated with either vehicle ( DMSO ) or MPZ ( 8 mg / kg ) daily i . p . , for 14 consecutive days , once tumors were palpable . Tumor volume was measured from the start of treatment until 1 week after treatment was removed . Graph of tumor volume on day 21 post - treatment is presented . Data are presented as the mean + s . e . m . n = 10 / group . Cryosections from GSC - xenografted tumors were stained for the endothelial marker PECAM1 ( red ) and tumor marker NESTIN ( green ) . Nuclei ( DAPI ) are shown in blue . Scale bar : 20 μm .

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

Figure 2Linear regression plot of in vitro limiting dilution assay (LDA) for GSC#9 treated with MALT1 inhibitor, mepazine (MPZ, 20 μM, 14 days). DMSO vehicle was used as a control. Data are representative of n=2.Stem cell frequency was calculated from LDA in GSCs #1, #4, and #12 treated with MPZ treatment (20 μM, 14 days). Data are presented as the mean + s.e.m. on 2 independent experiments.Tumorspheres per field of view (fov) were manually counted in GSCs #1, #4, #9, and #12 in response to MPZ (20 μM) and vehicle (DMSO), and in GSC#9 treated with Z-VRPR-FMK (75 µM) and vehicle (H2O) for 4 days. Data are presented as the mean + s.e.m. on 4 independent experiments for MPZ and 3 independent experiments for Z-VRPR-FMK.The expression of the stemness markers SOX2 and NESTIN was evaluated by western-blot and immunofluorescence (SOX2 in red NESTIN in green) in MPZ (+, 20 μM, 16 hours) and vehicle (-, DMSO, 16 hours) treated GSC#9. GAPDH served as a loading control. Scale bar: 10 μm.Cell viability was measured using Cell TiterGlo luminescent assay in GSCs #1, #4, #9, and #12, human brain endothelial cells (endo), human astrocytes (astro), and human neuron-like cells (neuron) treated for 48 hours with DMSO or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate.FACS analysis of EdU staining was performed on GSC#1 treated overnight with MPZ (10 μM). Data are presented as the mean + s.e.m. on 3 independent experiments.FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (20 μM).Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC#1 #4, and #9 (DGCs) treated for 48 hours with vehicle (DMSO) or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments. Morphology of GSCs #1, #4, #9 and DGCs #1, #4, #9 was shown using brightfield images.Heatmap of cell viability of GSC#9 using increasing doses (0, 5, 10, 20, 40 μM) of phenothiazines: mepazine (MPZ), fluphenazine (FLU), cyamemazine (CYAM), chlorpromazine (CHLO), pipotiazine (PIPO), alimemazine (ALI), promethazine (PRO), and doxylamine (DOXY). Data were normalized to their respective DMSO-treated controls.Nude mice were implanted with GSC#9 (106 cells) in each flank and randomized cages were treated with either vehicle (DMSO) or MPZ (8 mg/kg) daily i.p., for 14 consecutive days, once tumors were palpable. Tumor volume was measured from the start of treatment until 1 week after treatment was removed. Graph of tumor volume on day 21 post-treatment is presented. Data are presented as the mean + s.e.m. n=10/group.Cryosections from GSC-xenografted tumors were stained for the endothelial marker PECAM1 (red) and tumor marker NESTIN (green). Nuclei (DAPI) are shown in blue. Scale bar: 20 μm.


Figure 3Total protein lysates from GSCs # 1 and # 9 challenged with TNFα ( 10 ng / mL , for the indicated times ) were analyzed by western - blot for p - IκBα , IκBα and p - JNK . Total JNK and GAPDH served as loading controls . Western - blot analysis of p65 , cREL , and RELB in cytosolic ( cyt ) and nuclear ( nuc ) cell fractionation from GSC # 1 and GSC # 9 stimulated with TNFα ( 10 ng / mL , for the indicated times ) . TUBULIN and PARP served as controls for each fraction . Jurkat T - cells , GSC # 1 and GSC # 9 were stimulated with PMA ( 20 ng / mL ) and Ionomycin ( Iono , 300 ng / mL ) for 30 minutes . Total protein lysates were analyzed by western - blot for CYLD ( full length FL and cleaved c ' d ) , HOIL1 ( full length FL and cleaved c ' d ) , p - IκBα and IκBα . MALT1 and BCL10 served as loading controls . Jurkat T - cells , GSC # 1 and GSC # 9 were treated with vehicle ( DMSO ) and mepazine ( MPZ , 20 μM ) for 4 hours . PMA / Ionomycin mixture was also administered to Jurkat cells for the last 30 minutes . Total protein lysates were analyzed by western - blot for CYLD ( full length FL and cleaved c ' d ) . MALT1 served as a loading control . Western - blot analysis of CYLD ( full length , FL and cleaved , c ' d ) and MALT1 in total protein lysates from GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or MALT1 targeting duplexes ( siMALT1 ) . GAPDH served as a loading control . Densitometric analysis of c ' d CYLD / FL CYLD was performed ( right ) . Data are presented as the mean + s . e . m . on 5 independent experiments . ( Left ) Western - blot analysis of CYLD , ROQUIN1 / 2 , MALT1 , and BCL10 in total protein lysates from GSC # 9 treated for 4 hours with vehicle ( H2O ) or Z - VRPR - FMK ( 75 μM ) . GAPDH served as a loading control . ( Right ) Densitometric analysis of c ' d / FL was performed for ROQUIN1 / 2 and CYLD . Data are presented as the mean + s . e . m . on 3 independent experiments . Schematic drawing of MALT1 structures highlighting the C464A substitution in the protease - dead version . DD : Death Domain , C - like D : Caspase - like Domain , Ig : Immunoglobulin domain . Western - blot analysis of CYLD and FLAG in total protein lysates from GSC # 9 transfected with WT or C464A MALT1 - FLAG . GAPDH served as a loading control . Western - blot of CYLD ( full length FL and cleaved c ' d ) in total protein lysates from GSC # 9 after refreshing the medium ( + ) , as compared to 3 days - old culture ( - ) . GAPDH served as a loading control . Densitometric analysis of c ' d / FL CYLD was performed . Data are presented as the mean + s . e . m . on 5 independent experiments . Western - blot analysis of CYLD ( full length FL and cleaved c ' d ) in total protein lysates from GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or CARD10 targeting duplexes ( siCARD10 seq # 1 , seq # 2 , and seq # 3 ) . GAPDH served as a loading control . qPCR analysis confirmed the knock - down of CARD10 in GSC # 9 . Data are presented as the mean + s . e . m . on 3 independent experiments . Western - blot analysis of CYLD and BCL10 in total protein lysates from GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or BCL10 targeting duplexes ( siBCL10 , seq # 1 and seq # 3 ) . GAPDH served as a loading control . Cell viability was measured using Cell TiterGlo luminescent assay in sic and seq # 1 siBCL10 - transfected cells . Data were normalized to their respective sic - treated controls and are presented as the mean + s . e . m of 3 independent experiments , in triplicate .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Total protein lysates from GSCs #1 and #9 challenged with TNFα (10 ng/mL, for the indicated times) were analyzed by western-blot for p-IκBα, IκBα and p-JNK. Total JNK and GAPDH served as loading controls.Western-blot analysis of p65, cREL, and RELB in cytosolic (cyt) and nuclear (nuc) cell fractionation from GSC#1 and GSC#9 stimulated with TNFα (10 ng/mL, for the indicated times). TUBULIN and PARP served as controls for each fraction.Jurkat T-cells, GSC#1 and GSC#9 were stimulated with PMA (20 ng/mL) and Ionomycin (Iono, 300 ng/mL) for 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd), HOIL1 (full length FL and cleaved c'd), p-IκBα and IκBα. MALT1 and BCL10 served as loading controls.Jurkat T-cells, GSC#1 and GSC#9 were treated with vehicle (DMSO) and mepazine (MPZ, 20 μM) for 4 hours. PMA/Ionomycin mixture was also administered to Jurkat cells for the last 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd). MALT1 served as a loading control.Western-blot analysis of CYLD (full length, FL and cleaved, c'd) and MALT1 in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or MALT1 targeting duplexes (siMALT1). GAPDH served as a loading control. Densitometric analysis of c'd CYLD/FL CYLD was performed (right). Data are presented as the mean + s.e.m. on 5 independent experiments.(Left) Western-blot analysis of CYLD, ROQUIN1/2, MALT1, and BCL10 in total protein lysates from GSC#9 treated for 4 hours with vehicle (H2O) or Z-VRPR-FMK (75 μM). GAPDH served as a loading control. (Right) Densitometric analysis of c'd/FL was performed for ROQUIN1/2 and CYLD. Data are presented as the mean + s.e.m. on 3 independent experiments. Schematic drawing of MALT1 structures highlighting the C464A substitution in the protease-dead version. DD: Death Domain, C-like D: Caspase-like Domain, Ig: Immunoglobulin domain.Western-blot analysis of CYLD and FLAG in total protein lysates from GSC#9 transfected with WT or C464A MALT1-FLAG. GAPDH served as a loading control.Western-blot of CYLD (full length FL and cleaved c'd) in total protein lysates from GSC#9 after refreshing the medium (+), as compared to 3 days-old culture (-). GAPDH served as a loading control. Densitometric analysis of c'd/FL CYLD was performed. Data are presented as the mean + s.e.m. on 5 independent experiments.Western-blot analysis of CYLD (full length FL and cleaved c'd) in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or CARD10 targeting duplexes (siCARD10 seq#1, seq#2, and seq#3). GAPDH served as a loading control. qPCR analysis confirmed the knock-down of CARD10 in GSC#9. Data are presented as the mean + s.e.m. on 3 independent experiments.Western-blot analysis of CYLD and BCL10 in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or BCL10 targeting duplexes (siBCL10, seq#1 and seq#3). GAPDH served as a loading control. Cell viability was measured using Cell TiterGlo luminescent assay in sic and seq#1 siBCL10-transfected cells. Data were normalized to their respective sic-treated controls and are presented as the mean + s.e.m of 3 independent experiments, in triplicate.


Figure 4Transmission electron microscopy of GSC # 9 treated with vehicle ( DMSO ) or MPZ ( 20 μM ) for 16 hours . ER : endoplasmic reticulum ; MVB : multivesicular bodies ; lys : lysosome ; mit : mitochondria ; nuc : nucleus . Red stars denote lysosomes , blue stars vacuoles . Confocal analysis of LAMP2 staining ( red ) at 0 , 1 , 2 , 4 , and 6 hours post - MPZ ( 20 μM ) treatment . Nuclei ( DAPI ) are shown in blue . Scale bar : 10 μm . Western - blot analysis was performed in total protein lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or MALT1 targeting siRNA duplexes ( siMALT1 ) . Alternatively , western - blot analysis of LAMP2 , CTSD , and MALT1 was done in total protein lysates from GSC # 9 treated for 16 hours with MPZ ( 20 μM ) or Z - VRPR - FMK ( 75 µM ) . DMSO was used as vehicle . GAPDH served as a loading control . Confocal analysis of LAMP2 staining ( red ) in GSCs # 1 , # 4 , # 12 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) . Additionally , cells were transfected with non - silecing duplexes ( sic ) or MALT1 and BCL10 targeting siRNA duplexes ( siMALT1 and siBCL10 ) . Alternatively , lysotracker staining ( red ) was used to track for lysosomes in either GSC # 9 expressing either wild - type ( WT ) or C464A FLAG - MALT1 ( green ) . Scale bar : 10 μm . Quantification of LAMP2 staining pixel intensity on GSC # 9 treated as described in panel ( D ) . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 30 . Cryosections from GSC # 9 - xenografted tumors in vehicle and MPZ - challenged animals ( as described in Fig 2J ) and assessed for LAMP2 staining ( green ) . Nuclei ( DAPI ) are shown in blue . Scale bar : 10 μm . Western - blot analysis of LAMP2 was performed in tumor lysates . GAPDH served as a loading control . Confocal analysis of lysotracker staining ( red ) in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) ( upper panel ) or transfected with sic and siMALT1 ( bottom panel ) . As indicated , number of lysotracker - positive puncta and lysotracker pixel intensity ( arbitrary unit , AU ) were quantified per cell . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 30 . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm .

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

Figure 4Transmission electron microscopy of GSC#9 treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. ER: endoplasmic reticulum; MVB: multivesicular bodies; lys: lysosome; mit: mitochondria; nuc: nucleus. Red stars denote lysosomes, blue stars vacuoles.Confocal analysis of LAMP2 staining (red) at 0, 1, 2, 4, and 6 hours post-MPZ (20 μM) treatment. Nuclei (DAPI) are shown in blue. Scale bar: 10 μm.Western-blot analysis was performed in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Alternatively, western-blot analysis of LAMP2, CTSD, and MALT1 was done in total protein lysates from GSC#9 treated for 16 hours with MPZ (20 μM) or Z-VRPR-FMK (75 µM). DMSO was used as vehicle. GAPDH served as a loading control.Confocal analysis of LAMP2 staining (red) in GSCs #1, #4, #12 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM). Additionally, cells were transfected with non-silecing duplexes (sic) or MALT1 and BCL10 targeting siRNA duplexes (siMALT1 and siBCL10). Alternatively, lysotracker staining (red) was used to track for lysosomes in either GSC#9 expressing either wild-type (WT) or C464A FLAG-MALT1 (green). Scale bar: 10 μm.Quantification of LAMP2 staining pixel intensity on GSC#9 treated as described in panel (D). Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.Cryosections from GSC#9-xenografted tumors in vehicle and MPZ-challenged animals (as described in Fig 2J) and assessed for LAMP2 staining (green). Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis of LAMP2 was performed in tumor lysates. GAPDH served as a loading control.Confocal analysis of lysotracker staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (upper panel) or transfected with sic and siMALT1 (bottom panel). As indicated, number of lysotracker-positive puncta and lysotracker pixel intensity (arbitrary unit, AU) were quantified per cell. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.


Figure 5 ( Left ) Confocal analysis of LAMP2 ( red ) and LC3B ( green ) in GSC # 9 treated for 4 and 16 hours with vehicle ( DMSO ) and MPZ ( 20 µM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . ( Right ) Super - resolution imaging ( SIM , Structured Illumination Microscopy ) of LAMP2 ( red ) and LC3B ( green ) staining in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 µM ) . Western - blot analysis of LC3B and P62 in total protein lysates from GSC # 9 at 0 , 2 , 4 , 6 , and 16 hours post - MPZ treatment ( 20 µM ) . GAPDH served as a loading control . Western - blot analysis of LC3B in total protein lysates from GSCs # 1 and # 9 at 72 hours post - transfection with sic or siMALT1 , and subsequently treated 4 hours with vehicle ( DMSO ) or chloroquine ( CQ , 20 µM ) . Knockdown was verified by MALT1 blotting and GAPDH served as a loading control . Confocal analysis of LC3B ( green ) in GSC # 9 treated for16 hours with vehicle ( DMSO ) and MPZ ( 20 µM ) with or without chloroquine ( CQ , 20 µM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . GSC # 9 were transfected with LC3B reporters ( wild type WT or G120A mutant , which cannot be lipidated ) , treated 24 hours later with vehicle ( DMSO ) or MPZ ( 20 µM ) for 6 more hours . Ratios of WT / mutant luciferase signals are presented as the mean + s . e . m of 3 independent experiments . Confocal analysis of P62 staining ( red ) in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either transfected with sic or siMALT1 ( middle ) or treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) ( bottom ) . Quantification of P62 staining pixel intensity on GSC # 9 treated for 16 hours with vehicle ( DMSO or H2O ) , MPZ ( 20 µM ) or Z - VRPR - FMK ( 75 µM ) or sic and siMALT1 . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 30 . Cell viability was measuring using Cell TiterGlo in GSCs # 1 and # 9 pre - treated for 1 hour with vehicle ( DMSO ) or QVD ( 20 μM ) , and treated for 72 hours more with the indicated doses of MPZ . Data were normalized to the vehicle - treated controls and are presented as the mean + s . e . m of 4 independent experiments . FACS analysis of propidium iodide ( PI ) staining in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 15 μM ) in combination with QVD ( 20 μM ) . ( Left ) Percentage of PI positive cells , normalized to vehicle - treated controls are presented as the mean + s . e . m . on 3 independent experiments . ( Right ) Histogram plots for representative experiment ( DMSO in red and MPZ in blue ) . CSTD ELISA was performed on culture media from GSC # 9 treated for 8 hours with vehicle ( DMSO ) or MPZ ( 20 µM ) . Alternatively , cells were transfected with sic or siMALT1 and analyzed 72 hours later . Data are presented as the mean + s . e . m of 3 independent experiments . ( Left ) Cell viability was measured using Cell TiterGlo luminescent assay in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 10 μM ) , following a 30 minute - pre - treatment with the following drugs : Bafilomycin A1 ( Baf , 100 nM ) , Pepstatin A ( Pep , 1 μg / mL ) , or CTS inhibitor 1 ( Ctsi , 1 μM ) . Data were normalized to the vehicle - treated controls and are presented as the mean + s . e . m of 3 independent experiments in triplicate , stars refer to comparison to Vehicle + MPZ group ( blue squares ) . ( Right ) FACS analysis of propidium iodide ( PI ) staining in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 15 μM ) in combination with Baf , Pep and Ctsi . Percentage of PI positive cells normalized to vehicle - treated controls are presented as the mean + s . e . m . on 3 independent experiments .

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

Figure 5(Left) Confocal analysis of LAMP2 (red) and LC3B (green) in GSC#9 treated for 4 and 16 hours with vehicle (DMSO) and MPZ (20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. (Right) Super-resolution imaging (SIM, Structured Illumination Microscopy) of LAMP2 (red) and LC3B (green) staining in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 µM).Western-blot analysis of LC3B and P62 in total protein lysates from GSC#9 at 0, 2, 4, 6, and 16 hours post-MPZ treatment (20 µM). GAPDH served as a loading control.Western-blot analysis of LC3B in total protein lysates from GSCs #1 and #9 at 72 hours post-transfection with sic or siMALT1, and subsequently treated 4 hours with vehicle (DMSO) or chloroquine (CQ, 20 µM). Knockdown was verified by MALT1 blotting and GAPDH served as a loading control.Confocal analysis of LC3B (green) in GSC#9 treated for16 hours with vehicle (DMSO) and MPZ (20 µM) with or without chloroquine (CQ, 20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.GSC#9 were transfected with LC3B reporters (wild type WT or G120A mutant, which cannot be lipidated), treated 24 hours later with vehicle (DMSO) or MPZ (20 µM) for 6 more hours. Ratios of WT/mutant luciferase signals are presented as the mean + s.e.m of 3 independent experiments.Confocal analysis of P62 staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either transfected with sic or siMALT1 (middle) or treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (bottom). Quantification of P62 staining pixel intensity on GSC#9 treated for 16 hours with vehicle (DMSO or H2O), MPZ (20 µM) or Z-VRPR-FMK (75 µM) or sic and siMALT1. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.Cell viability was measuring using Cell TiterGlo in GSCs #1 and #9 pre-treated for 1 hour with vehicle (DMSO) or QVD (20 μM), and treated for 72 hours more with the indicated doses of MPZ. Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 4 independent experiments.FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with QVD (20 μM). (Left) Percentage of PI positive cells, normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments. (Right) Histogram plots for representative experiment (DMSO in red and MPZ in blue).CSTD ELISA was performed on culture media from GSC#9 treated for 8 hours with vehicle (DMSO) or MPZ (20 µM). Alternatively, cells were transfected with sic or siMALT1 and analyzed 72 hours later. Data are presented as the mean + s.e.m of 3 independent experiments.(Left) Cell viability was measured using Cell TiterGlo luminescent assay in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (10 μM), following a 30 minute-pre-treatment with the following drugs: Bafilomycin A1 (Baf, 100 nM), Pepstatin A (Pep, 1 μg/mL), or CTS inhibitor 1 (Ctsi, 1 μM). Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate, stars refer to comparison to Vehicle + MPZ group (blue squares). (Right) FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with Baf, Pep and Ctsi. Percentage of PI positive cells normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments.


Figure 6Heatmap of differentially expressed genes obtained from RNAseq analysis of GSC # 9 treated for 4 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) , from three biological replicates . Volcano plot of differentially expressed genes in RNAseq analysis of GSC # 9 , expressed as fold changes between vehicle ( DMSO ) and MPZ - treated cells . GSEA ( gene set enrichment analysis ) plot showing enrichment of " Bilanges serum and rapamycin sensitive genes " signature in vehicle ( DMSO ) versus MPZ - treated triplicates . qRT - PCR was performed on total RNA from GSC # 9 treated for 4 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Histograms showed changes in RNA expression of indicated targets . Data were normalized to two housekeeping genes ( ACTB , HPRT1 ) and are presented as the mean + s . e . m of technical triplicates . Western - blot analysis of LC3B , CSTD and TFEB in total protein lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or siRNA duplexes targeting TFEB ( siTFEB ) and treated with vehicle ( DMSO ) or MPZ ( 20 μM ) for 16 hours . GAPDH served as a loading control . Western - blot analysis of p - ULK1 , p - AKT , p - S6 , and p - p70S6K in GSC # 9 treated for 1 hour with MPZ ( 20 μM ) or rapamycin ( RAPA , 50 nM ) . Total ULK , AKT , S6 , and p70S6K served as loading controls . DMSO was used as a vehicle . Western - blot analysis of MALT1 , p - AKT , and p - S6 in total protein lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or MALT1 targeting siRNA duplexes ( siMALT1 ) . Total AKT and S6 , as well as GAPDH served as loading controls . Western - blot analysis of p - AKT , p - S6 , and p - p70S6K in total protein lysates from GSC # 9 treated for 1 hour with vehicle ( DMSO ) or 20 μM of phenothiazine compounds ( MPZ , FLU , CHLO , and CYA ) . Total AKT , total S6 and total p70S6K served as loading controls . Western - blot analysis of p - S6 and FLAG in GSC # 9 expressing WT or C464A MALT - FLAG . Total S6 and GAPDH served as loading controls . Confocal analysis of LAMP2 ( red ) and mTOR ( green ) staining in GSC # 9 treated with vehicle ( DMSO ) or MPZ ( 20 μM ) , Z - VRPR - FMK ( 75 μM ) , FLU ( 20 μM ) , CHLO ( 20 μM ) , and CYA ( 20 μM ) . Alternatively , cells were transfected with sic or siMALT1 . Nuclei ( DAPI ) are shown in blue . Arrows point to LAMP2 - positive area . Scale bars : 10 μm . Quantification of mTOR colocalization score with LAMP2 is shown . The Coloc2 plug - in from ImageJ was used to measure Mander ' s tM1 correlation factor in LAMP2 - positive ROI , using Costes threshold regression . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 10 .

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

Figure 6Heatmap of differentially expressed genes obtained from RNAseq analysis of GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM), from three biological replicates.Volcano plot of differentially expressed genes in RNAseq analysis of GSC#9, expressed as fold changes between vehicle (DMSO) and MPZ-treated cells.GSEA (gene set enrichment analysis) plot showing enrichment of "Bilanges serum and rapamycin sensitive genes" signature in vehicle (DMSO) versus MPZ-treated triplicates.qRT-PCR was performed on total RNA from GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM). Histograms showed changes in RNA expression of indicated targets. Data were normalized to two housekeeping genes (ACTB, HPRT1) and are presented as the mean + s.e.m of technical triplicates.Western-blot analysis of LC3B, CSTD and TFEB in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or siRNA duplexes targeting TFEB (siTFEB) and treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. GAPDH served as a loading control.Western-blot analysis of p-ULK1, p-AKT, p-S6, and p-p70S6K in GSC#9 treated for 1 hour with MPZ (20 μM) or rapamycin (RAPA, 50 nM). Total ULK, AKT, S6, and p70S6K served as loading controls. DMSO was used as a vehicle.Western-blot analysis of MALT1, p-AKT, and p-S6 in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Total AKT and S6, as well as GAPDH served as loading controls.Western-blot analysis of p-AKT, p-S6, and p-p70S6K in total protein lysates from GSC#9 treated for 1 hour with vehicle (DMSO) or 20 μM of phenothiazine compounds (MPZ, FLU, CHLO, and CYA). Total AKT, total S6 and total p70S6K served as loading controls.Western-blot analysis of p-S6 and FLAG in GSC#9 expressing WT or C464A MALT-FLAG. Total S6 and GAPDH served as loading controls.Confocal analysis of LAMP2 (red) and mTOR (green) staining in GSC#9 treated with vehicle (DMSO) or MPZ (20 μM), Z-VRPR-FMK (75 μM), FLU (20 μM), CHLO (20 μM), and CYA (20 μM). Alternatively, cells were transfected with sic or siMALT1. Nuclei (DAPI) are shown in blue. Arrows point to LAMP2-positive area. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.