( A ) ( B ) Pulsed - field gel electrophoresis ( PFGE ) analysis of wild type , mus81 ∆ , cdc5 - 2 and G2 - MUS81 cells in response to CPT . Cells were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , and released into S phase . Cells were collected at the indicated time points . DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE . Upper panel : agarose gel stained with ethidium bromide ; lower panel : Southern blot using a chromosome IV specific probe . JMs , joint molecules accumulated in the gel wells . The mean value of JMs relative to the total amount of DNA is indicated for each time point . Two to three biological replicates have been performed .

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(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.


( C ) ( D ) PFGE analysis of rad52 ∆ and rad52 ∆ mus81 ∆ cells in response to CPT performed as in ( A ) ( B ) . The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the + CPT 140 min time points in rad52 ∆ and rad52 ∆ mus81 ∆ cells are shown . Two biological replicates have been performed .

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(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.


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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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(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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


( A ) qRT - PCR analysis for Islr , Chd1 and Vim in total intestinal tissues , intestinal epithelium and lamina propria . n = 4 .

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(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4.


( B ) Western blotting for Islr and E - cadherin in total intestinal tissues , intestinal epithelium and lamina propria . α - Tubulin was used as a loading control .

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(B) Western blotting for Islr and E-cadherin in total intestinal tissues, intestinal epithelium and lamina propria. α-Tubulin was used as a loading control.


( C ) qRT - PCR analysis for Islr in CD45 + , CD45 - Epcam + , CD45 - Epcam - CD90 + and CD45 - Epcam - CD90 - cells sorted from intestinal tissues . n = 3 technical replicates .

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(C) qRT-PCR analysis for Islr in CD45+, CD45-Epcam+, CD45-Epcam-CD90+ and CD45-Epcam-CD90- cells sorted from intestinal tissues. n = 3 technical replicates.


( D ) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8 - week - old mice , showing that Islr is primarily expressed in stromal cells . Negative and positive controls were shown in Appendix Figure S1A . Scale bar : 25 μm .

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(D) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8-week-old mice, showing that Islr is primarily expressed in stromal cells. Negative and positive controls were shown in Appendix Figure S1A. Scale bar: 25 μm.


( E ) In situ hybridization for ISLR with RNAscope probe in human mucosa , inflamed mucosa from CD and UC patients . Scale bar : 25 μm .

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(E) In situ hybridization for ISLR with RNAscope probe in human mucosa, inflamed mucosa from CD and UC patients. Scale bar: 25 μm.


( F ) qRT - PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal . n = 4 at each time points .

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(F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points.


( G ) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3 - day or 5 - day DSS treatments . Scale bar : 50 μm .

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(G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm.


( H ) The cancer genome atlas ( TCGA ) RNA - seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues . The horizontal lines between the box limits represent the median , the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively . n = 65 .

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(H) The cancer genome atlas (TCGA) RNA-seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues. The horizontal lines between the box limits represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. n = 65.


( I ) Kaplan - Meier survival curve of 270 CRC cases . P = 0 . 02 ( log - rank test ) .

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(I) Kaplan-Meier survival curve of 270 CRC cases. P = 0.02 (log-rank test).


( J ) ISLR level increased with nodal metastasis in colorectal cancer patients . N0 , no regional lymph node metastasis ; N1 , metastases in 1 to 3 axillary lymph nodes ; N2 , metastases in 4 to 9 axillary lymph nodes ; N3 , metastases in 10 or more axillary lymph nodes . The solid horizontal lines represent the median , the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively .

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(J) ISLR level increased with nodal metastasis in colorectal cancer patients. N0, no regional lymph node metastasis; N1, metastases in 1 to 3 axillary lymph nodes; N2, metastases in 4 to 9 axillary lymph nodes; N3, metastases in 10 or more axillary lymph nodes. The solid horizontal lines represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively.


( K In situ hybridization for ISLR / Islr with RNAscope probes in normal human colon , human malignant adenocarcinoma , metastatic adenocarcinoma ( K ) , t indicating tumor ; a indicating adjacent tissues of tumor . Scale bar : 50 μm .

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(K In situ hybridization for ISLR/Islr with RNAscope probes in normal human colon, human malignant adenocarcinoma, metastatic adenocarcinoma (K), t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


L ) In situ hybridization for ISLR / Islr with RNAscope probes in mouse colon tumors from AOM - DSS model ( L ) . t indicating tumor ; a indicating adjacent tissues of tumor . Scale bar : 50 μm .

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L) In situ hybridization for ISLR/Islr with RNAscope probes in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


( B ) qRT - PCR analysis for Ets1 in colonic epithelium and lamina propria . n = 3 .

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(B) qRT-PCR analysis for Ets1 in colonic epithelium and lamina propria. n = 3.


( C ) Double immunofluorescence for ETS1 and β - catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients . Scale bar : 50 μm .

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(C) Double immunofluorescence for ETS1 and β-catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients. Scale bar: 50 μm.


( D ) qRT - PCR for Ets1 in colons with or without 5 - day DSS treatment . n = 4 .

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(D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4.


( E ) Double immunofluorescence for Ets1 and β - catenin in normal mouse colons without DSS treatment , mouse colons with 5 - day DSS treatments , and AOM - DSS mouse colon tumors . Scale bar : 50 μm .

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(E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm.


( F ) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA - seq ( P < 0 . 001 ; R = 0 . 68 ) .

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(F) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA-seq (P < 0.001; R = 0.68).


( G ) Western blotting for ISLR and HA in lysates of UC - MSCs transfected with pCMV5 - HA or pCMV5 - ETS1 . α - Tubulin was used as a loading control .

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(G) Western blotting for ISLR and HA in lysates of UC-MSCs transfected with pCMV5-HA or pCMV5-ETS1. α-Tubulin was used as a loading control.


( H ) Luciferase activity in lysates of UC - MSCs transfected with luciferase reporter plasmids of pGL3 - basic empty vector ( basic ) , wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions . n = 3 .

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(H) Luciferase activity in lysates of UC-MSCs transfected with luciferase reporter plasmids of pGL3-basic empty vector (basic), wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions. n = 3.


( I ) Chromatin immunoprecipitation assay was carried out on UC - MSCs cells using antibodies against ETS1 and Histone H3 . The antibody against Histone H3 was used as a positive control . IgG was used as a negative control . The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR . n = 3 technical replicates .

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(I) Chromatin immunoprecipitation assay was carried out on UC-MSCs cells using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of ETS1 binding to ISLR promoter was quantified using qPCR. n = 3 technical replicates.


( J ) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5 - day DSS treatment using antibodies against ETS1 and Histone H3 . The antibody against Histone H3 was used as a positive control . IgG was used as a negative control . The enrichment of Ets1 binding to Islr promoter was quantified using qPCR . n = 3 technical replicates .

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(J) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5-day DSS treatment using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of Ets1 binding to Islr promoter was quantified using qPCR. n = 3 technical replicates.


( A ) Quantification of body weight change in control ( n = 12 ) and cKO ( n = 13 ) mice after DSS treatment and DSS removal .

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(A) Quantification of body weight change in control (n=12) and cKO (n=13) mice after DSS treatment and DSS removal.


( B ) Fecal occult blood test in control and cKO mice at indicated timepoints . n = 3 at each timepoint .

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(B) Fecal occult blood test in control and cKO mice at indicated timepoints. n=3 at each timepoint.


( C ) Histological images of colonic tissues from control ( n = 10 ) and cKO ( n = 10 ) mice at indicated timepoints after DSS treatment . The Panels with non DSS are identical to Panels in Fig EV1F . Scale bar : 100 μm .

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(C) Histological images of colonic tissues from control (n=10) and cKO (n=10) mice at indicated timepoints after DSS treatment. The Panels with non DSS are identical to Panels in Fig EV1F. Scale bar: 100 μm.


( D ) Quantification of the clinical scores in control ( n = 12 ) and cKO ( n = 13 ) mice at indicated timepoints .

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(D) Quantification of the clinical scores in control (n=12) and cKO (n=13) mice at indicated timepoints.


( E ) Gross images of colons and quantification of colon length from control ( n = 6 ) and cKO ( n = 6 ) mice two days after 5 - day DSS treatment .

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(E) Gross images of colons and quantification of colon length from control (n=6) and cKO (n=6) mice two days after 5-day DSS treatment.


( F ) Double immunofluorescence for Ki67 and β - catenin in colons from control ( n = 4 ) and cKO ( n = 4 ) mice two days after 5 - day DSS treatment . Quantification of percentage of Ki67 + epithelial cells . Scale bar : 100 m .

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(F) Double immunofluorescence for Ki67 and β-catenin in colons from control (n=4) and cKO (n=4) mice two days after 5-day DSS treatment. Quantification of percentage of Ki67+ epithelial cells. Scale bar: 100 m.


( B ) Quantification of body weight changes in control ( n = 9 ) and cKO ( n = 10 ) mice during tumor development .

(B) Quantification of body weight changes in control (n=9) and cKO (n=10) mice during tumor development.


( C ) Gross images of AOM - DSS tumors in control ( n = 9 ) and cKO ( n = 10 ) mice .

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(C) Gross images of AOM-DSS tumors in control (n=9) and cKO (n=10) mice.


( D ) Quantification of tumor number per mouse and tumor volume in control ( n = 9 ) and cKO ( n = 10 ) mice .

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(D) Quantification of tumor number per mouse and tumor volume in control (n=9) and cKO (n=10) mice.


( E ) Histological images of colon tumors from control and cKO mice . Scale bar : 2 mm .

(E) Histological images of colon tumors from control and cKO mice. Scale bar: 2 mm.


( F Double immunofluorescence for Cyclin D1 and β - catenin ( F ) , in colon tumors from control ( n = 4 ) and cKO ( n = 4 ) mice . Scale bar : 100 μm .

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(F Double immunofluorescence for Cyclin D1 and β-catenin (F), in colon tumors from control (n=4) and cKO (n=4) mice. Scale bar: 100 μm.


G ) Double immunofluorescence for Ki67 and β - catenin ( G ) in colon tumors from control ( n = 4 ) and cKO ( n = 4 ) mice . Scale bar : 100 μm .

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G) Double immunofluorescence for Ki67 and β-catenin (G) in colon tumors from control (n=4) and cKO (n=4) mice. Scale bar: 100 μm.


( H ) Quantification of percentage of cyclin D1 + and Ki67 + cells in ( F and G ) . n = 4 .

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(H) Quantification of percentage of cyclin D1+ and Ki67+ cells in (F and G). n = 4.


( I ) The growth curve of HT29 CRC cells cultured in the supernatant with or without ISLR - HA . The supernatant were collected for HT29 CRC cells 24 hours after transfected with pcDNA3 . 1 empty vector or pcDNA3 . 1 - ISLR - HA vector . n = 5 at each timepoint .

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(I) The growth curve of HT29 CRC cells cultured in the supernatant with or without ISLR-HA. The supernatant were collected for HT29 CRC cells 24 hours after transfected with pcDNA3.1 empty vector or pcDNA3.1-ISLR-HA vector. n=5 at each timepoint.


( A ) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5 - day DSS treatment . The parameter of the color key indicated the fold changes converted to log2 of signal value normalized .

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(A) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5-day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized.


( B ) qRT - PCR analysis validates the altered Hippo - related genes in control and cKO mice . n = 4 .

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(B) qRT-PCR analysis validates the altered Hippo-related genes in control and cKO mice. n=4.


( C ) Western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5 - day DSS treatment . β - actin was used as a loading control . The quantification of pMst1 / 2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands .

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(C) Western blotting for pMst1/2, Mst1, pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands.


( D ) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5 - day DSS treatment . Histone H3 , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins . The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band .

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(D) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5-day DSS treatment. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band.


( E and F ) Double immunofluorescence for Yap1 and β - catenin in the colons from control ( n = 4 ) and cKO ( n = 4 ) mice two days after DSS removal ( E ) , and in AOM / DSS tumors from control ( n = 4 ) and cKO ( n = 4 ) mice ( F ) . The percentage of nuclear Yap1 + cells versus epithelial cells was quantified . Scale bar : 50 μm .

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(E and F) Double immunofluorescence for Yap1 and β-catenin in the colons from control (n=4) and cKO (n=4) mice two days after DSS removal (E), and in AOM/DSS tumors from control (n=4) and cKO (n=4) mice (F). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm.


( G ) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5 - day DSS treatment . β - actin was used as a loading control . The ratio of Yap1 / pYap1 were quantified . n = 3 .

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(G) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The ratio of Yap1/pYap1 were quantified. n=3.


( H ) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs , concomitantly transfected with PCDH empty vector or PCDH - YAP1 - 5SA vector . n = 4 technical replicates .

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(H) The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs, concomitantly transfected with PCDH empty vector or PCDH-YAP1-5SA vector. n = 4 technical replicates.


( I ) Pearson correlation analysis of ISLR and CTGF ( P < 0 . 001 ; R = 0 . 69 ) , ISLR and FSTL1 ( P < 0 . 001 ; R = 0 . 81 ) , as well as ISLR and CYR61 ( P < 0 . 001 ; R = 0 . 65 ) in human colorectal cancer based on TCGA RNA - seq database .

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(I) Pearson correlation analysis of ISLR and CTGF (P < 0.001; R = 0.69), ISLR and FSTL1 (P < 0.001; R = 0.81), as well as ISLR and CYR61 (P < 0.001; R = 0.65) in human colorectal cancer based on TCGA RNA-seq database.


( A ) Schematics of culture epithelial cells with supernatant from HEK293FT cells transfected with pcDNA3 . 1 empty vector or pcDNA3 . 1 - hISLR - HA plasmids . Western blotting for HA in the supernatant from HEK293FT cells transfected with the indicated plasmids .

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(A) Schematics of culture epithelial cells with supernatant from HEK293FT cells transfected with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for HA in the supernatant from HEK293FT cells transfected with the indicated plasmids.


( B ) Gene set enrichment for YAP / TAZ / TEAD direct target genes in transcriptome profiles for HEK293FT cells treated by hISLR - HA - enriched supernatant .

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(B) Gene set enrichment for YAP/TAZ/TEAD direct target genes in transcriptome profiles for HEK293FT cells treated by hISLR-HA-enriched supernatant.


( C ) Heatmap showing the altered Hippo related genes in the transcriptome profiles . The parameter of the color key indicated the fold changes converted to log2 of gene fragments per kilobase per million mapped reads ( FPKM ) values .

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(C) Heatmap showing the altered Hippo related genes in the transcriptome profiles. The parameter of the color key indicated the fold changes converted to log2 of gene fragments per kilobase per million mapped reads (FPKM) values.


( D ) Western blotting for pMST1 / 2 , MST1 , pMOB1 and MOB1 , pYAP1 and YAP1 in lysates from HEK293FT cells cultured in the supernatants with or without hISLR - HA . β - actin was used as a loading control .

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(D) Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from HEK293FT cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.


( E ) Western blotting for HA and ISLR in the supernatant from NCM460 colonic epithelial cells with pcDNA3 . 1 empty vector or pcDNA3 . 1 - hISLR - HA plasmids . Western blotting for pMST1 / 2 , MST1 , pMOB1 and MOB1 , pYAP1 and YAP1 in lysates from NCM460 CRC cells cultured in the supernatants with or without hISLR - HA . β - actin was used as a loading control .

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(E) Western blotting for HA and ISLR in the supernatant from NCM460 colonic epithelial cells with pcDNA3.1 empty vector or pcDNA3.1-hISLR-HA plasmids. Western blotting for pMST1/2, MST1, pMOB1 and MOB1, pYAP1 and YAP1 in lysates from NCM460 CRC cells cultured in the supernatants with or without hISLR-HA. β-actin was used as a loading control.


( F ) Immunofluorescence for YAP1 in HEK293FT cells cultured in the supernatant with or without hISLR - HA . Scale bar : 10 μm .

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(F) Immunofluorescence for YAP1 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. Scale bar: 10 μm.


( G ) Western blotting for YAP1 in cytoplasmic or nuclear proteins from HEK293FT cells cultured in the supernatant with or without hISLR - HA . Lamin a / c , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins .

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(G) Western blotting for YAP1 in cytoplasmic or nuclear proteins from HEK293FT cells cultured in the supernatant with or without hISLR-HA. Lamin a/c, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins.


( H ) qRT - PCR for CTGF and CYR61 in HEK293FT cells cultured in the supernatant with or without hISLR - HA . n = 3 .

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(H) qRT-PCR for CTGF and CYR61 in HEK293FT cells cultured in the supernatant with or without hISLR-HA. n=3.


( A ) Western blotting for Islr in the supernatant from primary cultured intestinal epithelial cells ( IECs ) transfected with pcDNA3 . 1 or pcDNA3 . 1 - mIslr - HA plasmids , and western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 , pYap1 and Yap1 in lysates from primary IECs cultured in the supernatants with or without mIslr - HA . β - actin was used as a loading control .

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(A) Western blotting for Islr in the supernatant from primary cultured intestinal epithelial cells (IECs) transfected with pcDNA3.1 or pcDNA3.1-mIslr-HA plasmids, and western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 in lysates from primary IECs cultured in the supernatants with or without mIslr-HA. β-actin was used as a loading control.


( B ) Western blotting for Yap1 in nuclear or cytoplasmic proteins from primary intestinal epithelial cells cultured in the supernatant with or without mIslr . Histone H3 , a positive control for nuclear proteins . α - Tubulin , a positive control for cytoplasmic proteins .

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(B) Western blotting for Yap1 in nuclear or cytoplasmic proteins from primary intestinal epithelial cells cultured in the supernatant with or without mIslr. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins.


( C ) qRT - PCR for Ctgf , Cry61 and Fstl1 in primary intestinal epithelial cells cultured in the supernatant with or without mIslr for 24 hours . n = 4 .

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(C) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal epithelial cells cultured in the supernatant with or without mIslr for 24 hours. n=4.


( D ) Activity of TEAD - Luciferase reporter in primary intestinal epithelial cells upon mIslr , Yap1 or mIslr and Yap1 . n = 4 .

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(D) Activity of TEAD-Luciferase reporter in primary intestinal epithelial cells upon mIslr, Yap1 or mIslr and Yap1. n=4.


( E ) The growth curve of primary intestinal epithelial cells upon mIslr or / and Yap1 siRNA over time . n = 5 .

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(E) The growth curve of primary intestinal epithelial cells upon mIslr or/and Yap1 siRNA over time. n=5.


( F ) qRT - PCR for Ctgf and Cry61 in primary intestinal epithelial cells 48 hours after treatments of mIslr or / and Yap1 siRNA ( siYap1 ) . n = 4 .

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(F) qRT-PCR for Ctgf and Cry61 in primary intestinal epithelial cells 48 hours after treatments of mIslr or/and Yap1 siRNA (siYap1). n=4.


( G ) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints . Scale bar : 100 μm . The organoids were co - cultured in a mixed medium of the supernatant with / without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1 : 4 . Quantification of organoids diameter at indicated timepoints . n > 150 organoids at each timepoint .

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(G) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints. Scale bar: 100 μm. The organoids were co-cultured in a mixed medium of the supernatant with/without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1:4. Quantification of organoids diameter at indicated timepoints. n>150 organoids at each timepoint.


( H ) Western blotting for pMst1 / 2 , Mst1 , pMob1 and Mob1 , pYap1 and Yap1 24 hours after the treatment . β - actin was used as a loading control .

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(H) Western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 24 hours after the treatment. β-actin was used as a loading control.


( I ) qRT - PCR for Ctgf , Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments . n = 3 .

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(I) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments. n=3.


( J ) Immunofluorescence for Yap1 and β - catenin in intestinal organoids 24 hours after the indicated treatments . The treatments are identical to ( G ) . The percentage of nuclear Yap1 + cells versus epithelial cells was quantified . Scale bar : 50 μm . n = 12 .

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(J) Immunofluorescence for Yap1 and β-catenin in intestinal organoids 24 hours after the indicated treatments. The treatments are identical to (G). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm. n=12.


B ) Western blot of the NOTCH3 EGF1 - 5 WT and R133C purified proteins . The eluate fractions were visualized by western blot using an α - myc antibody .

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B) Western blot of the NOTCH3 EGF1-5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α-myc antibody.


C ) Western blot of NOTCH3 EGF1 - 5 WT and R133C aggregated proteins . The incubated fractions of NOTCH3 EGF1 - 5 WT and R133C were visualized on a western blot using an α - myc antibody . The purified proteins and the aggregates were visualized after 1 - 5 days incubation by western blot using α - myc antibody under non - reducing conditions .

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C) Western blot of NOTCH3 EGF1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were visualized after 1-5 days incubation by western blot using α-myc antibody under non-reducing conditions.


A ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 7 months of age and TgN3R182C150 at 18 months of age . Representative images show brain arteries of TgN3R182C150 ( 7 and 18 months ) , sham and NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an α - SMA antibody ( green ) . Scale bar = 20µm . B ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) and non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of age . NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months ( n = 3 ) of age versus NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) and non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of age . Statistical significance was assessed using a Brown - Forsythe and Welch ANOVA tests followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel area ) : 7 m . o . vs . Sham , ns P = 0 . 9424 ; 7 m . o . vs . Vaccinated , ns P = 0 . 9809 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0046 ; Sham vs . Vaccinated , ns P = 0 . 9999 ; Sham vs . 18 m . o . * * P = 0 . 0032 ; Vaccinated vs . 18 m . o . * * P = 0 . 0032 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham , ns P = 0 . 7877 ; 7 m . o . vs . Vaccinated , ns P = 0 . 8358 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0069 ; Sham vs . Vaccinated , ns P = 0 . 9995 ; Sham vs . 18 m . o . * * P = 0 . 0044 ; Vaccinated vs . 18 m . o . * P = 0 . 0104 ) . NOTCH3 ECD deposits size : 7 m . o . vs . Sham , ns P = 0 . 1169 ; 7 m . o . vs . Vaccinated , ns P > 0 . 9999 ; 7 m . o . vs . 18 m . o . * * * P = 0 . 0009 ; Sham vs . Vaccinated , ns P = 0 . 0577 ; Sham vs . 18 m . o . * * P = 0 . 007 ; Vaccinated vs . 18 m . o . * * * * P < 0 . 0001 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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

A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). Scale bar =20µm. B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months (n=3) of age versus NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P=0.9424; 7 m.o. vs. Vaccinated, ns P=0.9809; 7 m.o. vs. 18 m.o.**P=0.0046; Sham vs. Vaccinated, ns P=0.9999; Sham vs. 18 m.o. **P=0.0032; Vaccinated vs. 18 m.o. **P=0.0032). NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham, ns P=0.7877; 7 m.o. vs. Vaccinated, ns P=0.8358; 7 m.o. vs. 18 m.o.**P=0.0069; Sham vs. Vaccinated, ns P=0.9995; Sham vs. 18 m.o. **P=0.0044; Vaccinated vs. 18 m.o. *P=0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P=0.1169; 7 m.o. vs. Vaccinated, ns P>0.9999; 7 m.o. vs. 18 m.o.***P=0.0009; Sham vs. Vaccinated, ns P=0.0577; Sham vs. 18 m.o. **P=0.007; Vaccinated vs. 18 m.o. ****P<0.0001, ns=non-significant). Error bars indicate standard error of the mean (SEM).