Significantly 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 ) .

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Significantly 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 .

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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 .

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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 ) .

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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 .

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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 ) .

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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).


Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr - or Ddx17 - depleted cells ( log 2 ratio scale ) .

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Heatmap 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 ) .

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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 .

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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.


qRT - 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 .

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qRT-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 ) .

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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 .

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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 .

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 .

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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 .

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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 .

Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.


Seven 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 ) .

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Seven 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 ) .

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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 .

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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 ) .

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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 .

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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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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).


( 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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(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.


( 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 ) ,

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(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 .

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(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 ) .

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(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).


( 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 )

(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 .

(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 ) .

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(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).


The 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 .

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The 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 .

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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 ) .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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.


Linear 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 .

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Linear 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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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.


Total 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 .

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Total 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 .

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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 .

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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 .

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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 .

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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 .

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(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 .

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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 .

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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 .

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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 .

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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.


Transmission 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 .

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Transmission 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 .

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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 .

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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 .

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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 .

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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 .

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

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.


( 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 ) .

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(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 .

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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 .

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

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 .

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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 .

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

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 .

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

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 .

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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 ) .

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

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 .

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

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

(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.


Heatmap 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 .

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Heatmap 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 .

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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 .

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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 .

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

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 .

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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 .

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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 .

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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 .

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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 .

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

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.


( Left ) Kaplan - Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level , using median cut - off , based on the TCGA RNAseq dataset . ( Right ) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function . Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes , together with 9 and 10 specific genes for MALT1 and QKI expression , respectively . ( Bottom ) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas ( TCGA , HG - U133A dataset ) on the GlioVis platform ( Bowman et al , 2007 ) . Pearson correlation factor = - 0 . 21 , p - value = 0 . 03 .

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(Left) Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level, using median cut-off, based on the TCGA RNAseq dataset. (Right) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function. Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes, together with 9 and 10 specific genes for MALT1 and QKI expression, respectively. (Bottom) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas (TCGA, HG-U133A dataset) on the GlioVis platform (Bowman et al, 2007). Pearson correlation factor=-0.21, p-value=0.03.


GSCs # 1 and # 9 protein lysates ( input ) were processed for immunoprecipitation ( IP ) using control immunoglobulins ( Ig ) , anti - QKI or anti - BCL10 antibodies . Input and IP fractions were separated on SDS - PAGE and western - blots for MALT1 , QKI and BCL10 antibodies were performed as specified .

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GSCs #1 and #9 protein lysates (input) were processed for immunoprecipitation (IP) using control immunoglobulins (Ig), anti-QKI or anti-BCL10 antibodies. Input and IP fractions were separated on SDS-PAGE and western-blots for MALT1, QKI and BCL10 antibodies were performed as specified.


Total protein lysates ( input ) from GSC # 9 treated with vehicle ( - , DMSO ) or MPZ ( + , 20 μM , 1 hour ) or with vehicle ( - , H2O ) or Z - VRPR - FMK ( + , 75 μM , 4 hours ) , were processed for control immunoglobulins ( Ig ) or anti - QKI antibodies immunoprecipitation ( IP ) . Western - blots were performed with indicated antibodies . Western - blots were performed with indicated antibodies .

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

Total protein lysates (input) from GSC#9 treated with vehicle (-, DMSO) or MPZ (+, 20 μM, 1 hour) or with vehicle (-, H2O) or Z-VRPR-FMK (+, 75 μM, 4 hours), were processed for control immunoglobulins (Ig) or anti-QKI antibodies immunoprecipitation (IP). Western-blots were performed with indicated antibodies. Western-blots were performed with indicated antibodies.


Confocal analysis of Lysotracker ( green ) or FLAG ( red ) in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue .

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Confocal analysis of Lysotracker (green) or FLAG (red) in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue.


Confocal analysis of LAMP2 ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue . Quantification of LAMP2 staining pixel intensity on GSC # 9 transfected with mock and FLAG - QKI . Data are presented as the mean + s . e . m . on 3 independent experiments . Each dot represents one cell . n > 15 .

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Confocal analysis of LAMP2 (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Quantification of LAMP2 staining pixel intensity on GSC#9 transfected with mock and FLAG-QKI. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>15.


Western - blot analysis of QKI , LAMP2 and LC3B in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . GAPDH served as a loading control .

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Western-blot analysis of QKI, LAMP2 and LC3B in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. GAPDH served as a loading control.


Confocal analysis of mTOR ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or Flag - QKI . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm .

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Confocal analysis of mTOR (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or Flag-QKI. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.


GSC # 1 were transfected with either empty vector ( mock ) or FLAG - QKI . Total protein lysates were processed for western - blots against p - S6 and FLAG . Total S6 served as a loading control .

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GSC#1 were transfected with either empty vector (mock) or FLAG-QKI. Total protein lysates were processed for western-blots against p-S6 and FLAG. Total S6 served as a loading control.


Fraction of surviving cells over time in GSCs # 1 and # 9 , transduced with empty vector ( mock ) or FLAG - QKI bi - cistronic GFP plasmids . 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 ) . Data are representative of n = 3 .

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Fraction of surviving cells over time in GSCs #1 and #9, transduced with empty vector (mock) or FLAG-QKI bi-cistronic GFP plasmids. 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). Data are representative of n=3.


GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 16 hours with vehicle ( DMSO ) or MPZ ( 10 μM ) . Total protein lysates were processed for western - blots against LAMP2 , CTSD , QKI , and LC3B expression , as indicated . GAPDH served as a loading control .

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

GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 16 hours with vehicle (DMSO) or MPZ (10 μM). Total protein lysates were processed for western-blots against LAMP2, CTSD, QKI, and LC3B expression, as indicated. GAPDH served as a loading control.


Confocal analysis of mTOR ( green ) and LAMP2 ( red ) in GSC # 9 transfected with sic or siQKI , and treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Nuclei ( DAPI ) are shown in blue . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. 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.


GSC # 9 transfected with non - silencing RNA duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 1 hour with vehicle ( DMSO ) or MPZ ( 20 μM ) . Total protein lysates were processed for western - blots against QKI and p - S6 . TUBULIN and Total S6 served as loading controls .

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

GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 1 hour with vehicle (DMSO) or MPZ (20 μM). Total protein lysates were processed for western-blots against QKI and p-S6. TUBULIN and Total S6 served as loading controls.


FACS analysis of EdU staining was performed on GSC # 9 cells transfected with non - silencing RNA duplexes ( sic , pink ) , QKI targeting siRNA duplexes ( siQKI , light purple ) , MALT1 targeting siRNA duplexes ( siMALT1 , blue ) or double transfected with siQKI and siMALT1 ( purple ) .

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FACS analysis of EdU staining was performed on GSC#9 cells transfected with non-silencing RNA duplexes (sic, pink), QKI targeting siRNA duplexes (siQKI, light purple), MALT1 targeting siRNA duplexes (siMALT1, blue) or double transfected with siQKI and siMALT1 (purple).


FACS analysis of propidium iodide ( PI ) staining in GSC # 9 transfected with non - silencing RNA duplexes ( sic ) , QKI targeting siRNA duplexes ( siQKI ) , MALT1 targeting siRNA duplexes ( siMALT1 ) or double transfected with siQKI and siMALT1 and analyzed 72 hours later . Percentage of PI - positive cells normalized to vehicle treated controls are presented as the mean + s . e . m . on 3 independent experiments .

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FACS analysis of propidium iodide (PI) staining in GSC#9 transfected with non-silencing RNA duplexes (sic), QKI targeting siRNA duplexes (siQKI), MALT1 targeting siRNA duplexes (siMALT1) or double transfected with siQKI and siMALT1 and analyzed 72 hours later. Percentage of PI-positive cells normalized to vehicle treated controls are presented as the mean + s.e.m. on 3 independent experiments.


B . Chromatin accessibility and p300 binding at differential ATAC - seq peaks visualized using K - means clustering .

B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.


B . Boxplots showing differential gene expression ( AC versus DMZ ) and associated ATAC - seq signals ( Two biological replicates ) . The central band within the boxplot represents the median and the whiskers show 1 . 5 times the range between the first and third quartile ( IQR ) .

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B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR).


F . Genomic tracks showing aggregated accessibility of single - cell ATAC - seq clusters at the t ( tbxt ) , tfap2a , gsc , ctcf , lhx1 and sox11 loci .

F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.


C . Feature maps , showing the expression of selected genes in single cells ( cf . Appendix Fig . S2 ) . Color scale represents the gene expression value ( log1p - transformed counts per 10 , 000 unique reads ) for each cell for a given gene , from low ( grey ) to high ( orange ) .

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C. Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig. S2). Color scale represents the gene expression value (log1p-transformed counts per 10,000 unique reads) for each cell for a given gene, from low (grey) to high (orange).


A . Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1 , Foxb1 - Eomes , Eomes , Irx3 , Irx3 - Otx2 , Otx2 and Lhx8 .

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A. Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1-Eomes, Eomes, Irx3, Irx3-Otx2, Otx2 and Lhx8.


C . Fold enrichment of genes with zygotically defined ( ZyD ) H3K4me3 at their promoter in AC with transcription factor overexpression . Asterisk indicates hypergeometric p - value < = 0 . 01 .

C. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric p-value <= 0.01.


B ) Expression of the 21U sensor ( left ) and DAPI staining ( right ) of gonad arms in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . Number of animals analysed and with indicated phenotype are given in the panel . Animals not showing the activation of the 21U sensor ( + ) were still silenced , and animals that did not show the silenced 21U sensor ( RNAe ) state were expressing weakly , comparable to the 21U sensor ( + ) . Scale bar : 25 μm .

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B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.


C ) Tc1 reversion assay in different genetic backgrounds . All the strains tested carried the unc - 22 : : Tc1 ( st136 ) allele . Tc1 excision can result in restoration of unc - 22 function , which can be scored visually . Negative control = unc - 22 : : Tc1 ( st136 ) in a wild type background ; positive control = wago - 1 / - 2 / - 3 . Two independent experiments per strain are represented .

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C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented.


D ) Crossing scheme to address the re - initiation of silencing of the 21U sensor . A mut - 7 mutant male expressing the 21U sensor is crossed with either a wild type ( left ) , prg - 1 ( middle ) or pid - 2 ( right ) mutant hermaphrodite . Their F1 offspring was scored for expression of the 21U sensor by microscopy . Three states of expression were scored , and represented in a pie chart . The three expression states are exemplified by representative images at the bottom : OFF ( left ) , FAINT ( right ) or ON ( middle ) . DIC images are shown above the fluorescence panels . Gonads are outlined by a dashed line . Scale bar : 25 µm .

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D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm.


E ) Crossing scheme to address if maternal 21U RNAs are sufficient to re - initiate the silencing of the 21U sensor . A pid - 1 mutant male expressing the 21U sensor is crossed with a hermaphrodite , heterozygous for the same mutation . All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite , but in 50 % of the F1 , which is pid - 1 homozygous mutant , no zygotic PID - 1 is present , hence no zygotic 21U RNAs can be made . The silencing or expression of the 21U sensor in the pid - 1 homozygous mutant F1 has been scored by microscopy , and depicted in a pie chart . At the bottom , a representative image of an animal carrying a silenced 21U sensor ( lower : mCherry signal ; upper : DAPI staining ) in pid - 1 mutant offspring . Gonads are outlined by a dashed line . Scale bar : 25 μm .

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E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.


F ) Crossing scheme to test the role of PID - 2 in re - initiating the silencing of the 21U sensor , mediated by maternally provided 21U RNAs only . The expression of the 21U sensor in the F1 has been scored by microscopy , and depicted in a pie chart . White arrow heads indicate the many arrayed oocytes , typical of a feminized germline . DIC and fluorescence image of a representative animal are shown at the bottom . Gonads are outlined by a dashed line . Scale bar : 25 μm .

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F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.


The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data , and read density was plotted over the transgene , which is schematically depicted at the bottom . The aggregated results of three replicates is shown for each indicated genotype in the different panels . The shading , in grey , represents the standard deviation of the read density from the three replicates . ( + ) means that the sensor was detectably expressed . ( RNAe ) means that the sensor was crossed into the respective mutant background in an RNAe state ( i . e . its silencing was PRG - 1 - independent ) , and its expression remained undetectable by microscopy .

The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data, and read density was plotted over the transgene, which is schematically depicted at the bottom. The aggregated results of three replicates is shown for each indicated genotype in the different panels. The shading, in grey, represents the standard deviation of the read density from the three replicates. (+) means that the sensor was detectably expressed. (RNAe) means that the sensor was crossed into the respective mutant background in an RNAe state (i.e. its silencing was PRG-1-independent), and its expression remained undetectable by microscopy.


A ) MA - plot of log2 fold changes ( Y - axis ) versus the mean of normalized counts of 22G RNAs ( X - axis ) for pid - 2 ( xf23 ) mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 .

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A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


B ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 2 ( xf23 ) mutants . Significance was tested with Fisher ' s exact test and p - values adjusted with the Benjamini - Hochberg Procedure . Colour scheme represents the odds ratio of overlaps representing the strength of association . N . S . : not significant .

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B) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.


C - D ) Cumulative 22G coverage along the gene body of Mutator and CSR - 1 targets . Values represent 22G coverage normalized to the total coverage of each gene , for wild type ( N2 ) and pid - 2 ( xf23 ) mutants . The lines represent the average of biological replicates , whereas the shading represents the standard deviation of biological replicates . a . u . : arbitrary units .

C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.


A ) Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . As control , eGFP IPs were performed on protein extracts from wild - type , non - transgenic animals . For each IP - MS experiment , quadruplicates were measured and analysed . The dashed line reflects a significance threshold of p - value < 0 . 05 at two - fold enrichment .

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A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.


Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . In this experiment , the endogenous PID - 2 protein was immunoprecipitated , and pid - 2 ( xf23 ) mutant protein extracts were used as control .

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Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.


Volcano plot , representing the enrichment of proteins interacting with PID - 4 , by immunoprecipitating endogenously tagged PID - 4 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control .

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Volcano plot , representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


Volcano plot , representing the enrichment of proteins interacting with PID - 5 , by immunoprecipitating endogenously tagged PID - 5 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control .

Volcano plot, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


A ) Expression of the 21U sensor in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . The panels on the right are DIC images of the respective animals . Scale bar : 25 μm .

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A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.


B ) 22G RNA coverage of the 21U sensor ( RNAe ) in the indicated genetic backgrounds .

B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds.


C ) MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) for pid - 4 mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 .

C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


D ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 4 mutants . Colour scheme represents the odds ratio of overlaps representing the strength of association .

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D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.


MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . E ) for pid - 5

MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutant


Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in F ) pid - 5

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Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutant


MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . G ) for pid - 4 ; pid - 5 double

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MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutant


Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in H ) pid - 4 ; pid - 5 double

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Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutant


I , J ) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC . Six L2 - L3 larvae for each of the indicated backgrounds were hand - picked to a fresh plate every four or six days , counting as two or three generations , respectively , until no larvae were present on the plate to be picked . * : two plates of N2 were contaminated and were excluded from the assay .

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I, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.


Expression of PID - 2 : : eGFP ( xfSi145 ) ( A ) in perinuclear granules in the germline . PGL - 1 : : mTagRFP - T ( A ) mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm .

Expression of PID-2::eGFP(xfSi145) (A) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of , PID - 4 : : mTagRFP - T ( B ) in perinuclear granules in the germline . DEPS - 1 : : GFP ( B mark P granules . The indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize the granules , and their overlaps . One L4 gonad is shown for each animal . Note that most of the L4 gonad is in pachytene stage . Arrow heads indicate individual condensates . Scale bar : 25 µm .

Expression of , PID-4::mTagRFP-T (B) in perinuclear granules in the germline. DEPS-1::GFP (B mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.