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

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

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


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

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

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


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

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

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


Figure 7 ( 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 . 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 . 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 . 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 . 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 . 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 . 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 .

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

Figure 7(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.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.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.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.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.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.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.


Figure 1 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 1(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.(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.(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.(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.(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.(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.


Figure 2 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 2(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.(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.(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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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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Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 . ( 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 . ( 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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Figure 4(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.(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.(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 5(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.(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.(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.(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.


Figure 6 ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 6(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.(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.(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.(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.


Figure 7 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 7(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.(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.(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.(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.(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.


Figure 2B ) 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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Figure 2B) 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.


Figure 4A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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).


Figure 5A ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 3 , 7 and 18 months of age . Representative images show brain arteries and capillaries of TgN3R182C150 , sham and NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an anti - perlecan 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 a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non - vaccinated 3 months old TgN3R182C150 ( n = 3 ) and 7 months old TgN3R182C150 ( n = 6 ) mice and 18 months old TgN3R182C150 ( n = 3 ) . Quantification of NOTCH3 - ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 - ECD stained area and average size per vessel revealed a significant decrease in NOTCH3 - ECD deposition in brain arteries and capillaries between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice . 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 ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0029 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0004 ; 7 m . o . vs . Sham , ns P = 0 . 7326 ; 7 m . o . vs . Vaccinated , * * P = 0 . 003 ; Sham vs . Vaccinated , * * * * P < 0 . 0001 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0038 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0002 ; 7 m . o . vs . Sham , ns P = 0 . 9913 ; 7 m . o . vs . Vaccinated , * P = 0 . 021 ; Sham vs . Vaccinated , * * * P = 0 . 001 ) . NOTCH3 ECD deposits size : 3 m . o . vs . 18 m . o . * P = 0 . 0215 ; 3 m . o . vs . 7 m . o . * * P = 0 . 0029 ; 7 m . o . vs . Sham , ns P = 0 . 3622 ; 7 m . o . vs . Vaccinated , * P = 0 . 0428 ; Sham vs . Vaccinated , * * P = 0 . 0038 , ns = non - significant ) . Dotted lines indicate quartiles and dashed thicker lines are the median .

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

Figure 5A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan 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 a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C150 (n=3) and 7 months old TgN3R182C150 (n=6) mice and 18 months old TgN3R182C150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF1-5 -immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice. 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): 3 m.o. vs. 18 m.o. **P=0.0029; 3 m.o. vs. 7 m.o. ***P=0.0004; 7 m.o. vs. Sham, ns P=0.7326; 7 m.o. vs. Vaccinated, **P=0.003; Sham vs. Vaccinated, ****P<0.0001). NOTCH3 ECD deposits (number/1000 um2): 3 m.o. vs. 18 m.o. **P=0.0038; 3 m.o. vs. 7 m.o. ***P=0.0002; 7 m.o. vs. Sham, ns P=0.9913; 7 m.o. vs. Vaccinated, *P=0.021; Sham vs. Vaccinated, ***P=0.001). NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. *P=0.0215; 3 m.o. vs. 7 m.o. **P=0.0029; 7 m.o. vs. Sham, ns P=0.3622; 7 m.o. vs. Vaccinated, *P=0.0428; Sham vs. Vaccinated, **P=0.0038, ns= non-significant). Dotted lines indicate quartiles and dashed thicker lines are the median.


Figure 6A ) NOTCH3 ECD was detected in the whole blood serum of the non - treated TgN3R182C150 mice at three months ( n = 9 ) of age and further increased at seven months ( n = 6 ) of age . Serum from Notch3 - / - and C57Bl6 / J WT mice were included as negative controls . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( 3 m . o . vs 7 m . o . * * * P = 0 . 0005 ) . Error bars indicate standard error of the mean ( SEM ) . B ) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated ( n = 10 ) TgN3R182C150 mice vs sham ( n = 9 ) . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( Sham vs Vaccinated * P = 0 . 0196 ) . Error bars indicate standard error of the mean ( SEM ) .

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

Figure 6A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C150 mice at three months (n=9) of age and further increased at seven months (n=6) of age. Serum from Notch3 -/- and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired student t test with Welch's correction. (3 m.o. vs 7 m.o. ***P=0.0005). Error bars indicate standard error of the mean (SEM).B) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated (n=10) TgN3R182C150 mice vs sham (n=9). Statistical analysis was performed using unpaired student t test with Welch's correction. (Sham vs Vaccinated *P=0.0196). Error bars indicate standard error of the mean (SEM).


Figure 7A ) Representative images show microglia stained with anti - CD68 antibody ( red ) and Iba1 antibody ( green ) . Scale bar = 20µm . B ) Quantification of CD68 - stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1 - 5 - immunized ( n = 6 ) , sham ( n = 4 ) and non - vaccinated TgN3R182C150 ( n = 5 ) mice . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with CD68 staining : 7 m . o . vs . Sham ns P = 0 . 7393 ; Sham vs . Vaccinated * * P = 0 . 0087 . CD68 staining ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 9131 ; Sham vs . Vaccinated * P = 0 . 0157 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) . C ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an antibody against microglia ( Iba1 , green ) . Scale bar = 20µm . D ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice at 7 months of age . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with NOTCH3 ECD deposits : 7 m . o . vs . Sham ns P = 0 . 7997 ; Sham vs . Vaccinated ns P = 0 . 0526 . NOTCH3 ECD deposits ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 5362 ; Sham vs . Vaccinated ns P = 0 . 8059 . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham ns P = 0 . 8392 ; Sham vs . Vaccinated ns P = 0 . 5777 . NOTCH3 ECD deposits size : 7 m . o . vs . Sham ns P = 0 . 6664 ; Sham vs . Vaccinated ns P = 0 . 8787 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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

Figure 7A) Representative images show microglia stained with anti-CD68 antibody (red) and Iba1 antibody (green). Scale bar=20µm. B) Quantification of CD68-stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1-5-immunized (n=6), sham (n=4) and non-vaccinated TgN3R182C150 (n=5) mice. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with CD68 staining: 7 m.o. vs. Sham ns P=0.7393; Sham vs. Vaccinated **P=0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P=0.9131; Sham vs. Vaccinated *P=0.0157, ns=non-significant). Error bars indicate standard error of the mean (SEM).C) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar=20µm. D) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5-immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice at 7 months of age. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P=0.7997; Sham vs. Vaccinated ns P=0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P=0.5362; Sham vs. Vaccinated ns P=0.8059. NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham ns P=0.8392; Sham vs. Vaccinated ns P=0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P=0.6664; Sham vs. Vaccinated ns P=0.8787, ns=non-significant). Error bars indicate standard error of the mean (SEM).


Figure 1C - F Division pattern of seam cells at the postembryonic stage in hermaphrodites . Seam cells V1 - V4 and V6 divide symmetrically at the L2 stage ( highlighted in red ) , thus doubling in number . ( G , H ) dcr ‐ 1 young adults have an increased number of seam cells ( shown in brackets ) and gaps in the alae ( between arrows ) . ( I , J ) atg ‐ 2 mutations suppress the retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants . K - M Confocal images showing expression of hbl ‐ 1 : : gfp : : hbl ‐ 1 reporter in VNC in wild type ( K ) , atg ‐ 2 ( L ) and epg ‐ 6 ( M ) L3 larvae . N Distribution of relative fluorescence intensity of HBL ‐ 1 : : GFP in 50 unit areas in VNC in the indicated strains . O - Q Confocal images showing the expression of the col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) reporter in wild type ( O ) , atg ‐ 2 ( P ) and atg ‐ 3 ( Q ) late L4 larvae . R Distribution of relative fluorescence intensity of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal cells in the indicated strains . S , T Distribution of relative fluorescence intensity of hbl ‐ 1 : : gfp in VNC and col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal cells in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated animals . Black lines in ( N , R - T ) represent the average fluorescence intensity of the reporter in each strain . P values are determined by Student ' s two ‐ tailed unpaired t ‐ test in ( N , R - T ) . ( C , E , G , I ) Scale bar , 20 μm ; ( D , F , H , J - M , O - Q ) scale bar , 10 μm . GFP , green fluorescent protein ; miRISC , miRNA ‐ induced silencing complex ; RNAi , RNA ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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

Figure 1C-F Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number.(G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows).(I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants.K-M Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae.N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains.O-Q Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae.R Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains.S,T Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R-T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R-T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J-M,O-Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.


Figure 2A - C Wild ‐ type and atg ‐ 2 hermaphrodites have a single vulva ( arrow ) . ( B ) let ‐ 60 ( n1046gf ) mutants show a Muv phenotype . Black arrows indicate pseudovulvae . D Loss of autophagy activity suppresses the Muv phenotype in let ‐ 60 ( n1046 ) mutants . The number of adult animals examined was : wild type ( n = 450 ) , let ‐ 60 ( n = 406 ) , epg ‐ 6 ; let ‐ 60 ( n = 444 ) , let ‐ 60 ; atg ‐ 2 ( n = 803 ) , epg ‐ 5 ; let ‐ 60 ( n = 672 ) , epg ‐ 5 ; let ‐ 60 ; ain ‐ 1 ( ku322 ) ( n = 635 ) . P values are determined by a two ‐ tailed χ2 test . E , F lsy ‐ 6 mutants show defects in ASEL fate specification and do not express the ASEL ‐ specific reporter lim ‐ 6pro : : GFP . G Expression of lim ‐ 6pro : : GFP in various strains . aCompared with lsy ‐ 6 ( ot150 ) . bCompared with lsy ‐ 6 ( ot150 ) ; ain ‐ 1 ( ku322 ) . P values are determined by a two ‐ tailed χ2 test .

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

Figure 2A-C Wild‐type and atg‐2 hermaphrodites have a single vulva (arrow). (B) let‐60(n1046gf) mutants show a Muv phenotype. Black arrows indicate pseudovulvae.D Loss of autophagy activity suppresses the Muv phenotype in let‐60(n1046) mutants. The number of adult animals examined was: wild type (n=450), let‐60 (n=406), epg‐6; let‐60 (n=444), let‐60; atg‐2 (n=803), epg‐5; let‐60 (n=672), epg‐5; let‐60; ain‐1(ku322) (n=635). P values are determined by a two‐tailed χ2 test.E,F lsy‐6 mutants show defects in ASEL fate specification and do not express the ASEL‐specific reporter lim‐6pro::GFP.G Expression of lim‐6pro::GFP in various strains. aCompared with lsy‐6(ot150).bCompared with lsy‐6(ot150); ain‐1(ku322). P values are determined by a two‐tailed χ2 test.


Figure 3AIN ‐ 1 : : GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild ‐ type animals . ( A ) Nomarski image of the embryo shown in ( B ) . Scale bar , 10 μm for whole embryo . C . elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size . Thus , scale bars for embryos are only shown once in each figure . ( C ) AIN ‐ 1 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos . ( A - C ) are confocal images . ( D ) Western blotting shows that endogenous AIN ‐ 1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type . ( E ) No AIN ‐ 1 : : GFP aggregates are formed in sqst ‐ 1 mutant embryos . ( F ) AIN ‐ 1 : : GFP accumulates into numerous aggregates in epg ‐ 7 mutant embryos . ( G , H ) Number of AIN ‐ 1 : : GFP ( G ) and ALG ‐ 2 : : GFP ( H ) aggregates per focal plane in the indicated strains . Error bars indicate the s . d . of four examined embryos . ( I , J ) ALG ‐ 2 : : GFP is diffusely localized in the cytoplasm in wild ‐ type embryos . ( I ) Nomarski image of the embryo shown in ( J ) . ( K ) ALG ‐ 2 : : GFP forms a few aggregates in epg ‐ 6 mutant embryos . ( L ) ALG ‐ 2 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos under stress conditions . GFP , green fluorescent protein ; s . d . , standard deviation .

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Figure 3AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A-C) are confocal images.(D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type.(E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos.(F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos.(G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos.(I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J).(K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.


Figure 4 ( A - D ) Both AIN ‐ 1 : : GFP and SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed and diffusely localized in wild ‐ type embryos . ( E - H ) AIN ‐ 1 : : GFP aggregates colocalize with SQST ‐ 1 aggregates in atg ‐ 3 mutant embryos . Scale bars , 10 μm for insets that show a magnified view . ( I - L ) AIN ‐ 1 : : GFP aggregates are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant embryos . ( M - P ) Colocalization of AIN ‐ 1 : : GFP aggregates with LGG ‐ 1 puncta in epg ‐ 6 mutant embryos . ( Q ) % of AIN ‐ 1 : : GFP aggregates colocalized with SQST ‐ 1 aggregates , SEPA ‐ 1 aggregates and LGG ‐ 1 puncta in autophagy mutants . Error bars indicate the s . d . of four examined embryos . ( R ) A co ‐ IP assay shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo . ( S ) Direct interaction between AIN ‐ 1 and LGG ‐ 1 in an in vitro pull ‐ down assay . 5 % of the His fusion proteins used for pull ‐ down are shown as input . Corresponding Ponceau ‐ stained blots show the amount of proteins used for pull ‐ down . ( T ) A fragment of AIN ‐ 1 directly binds to LGG ‐ 1 in an in vitro pull ‐ down assay .

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

Figure 4(A-D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos.(E-H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view.(I-L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos.(M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos.(Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos.(R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo.(S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down.(T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay.


figf1 ( a ) Knockdown of dRagA and dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 cells untreated ( lane 2 ) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies . ( b ) dRagA and dRagC are not required for dAkt phosphorylation . Drosophila S2 cells untreated ( lanes 1 - 3 ) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min . Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated . NC , negative control RNAi . ( c ) dRagA and dRagC function in parallel with TSC2 and PTEN . dRagA and / or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) and this increase was compromised by dRagA or / and dRagC RNAi . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

figf1(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies.(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi.(c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf2 ( a ) Constitutively active RagA and RagB stimulate S6K phosphorylation . 200 ng of each mammalian RagA , RagB , RagC or RagD construct was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) and constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( b ) RagA has a dominant role over RagC in regulating S6K phosphorylation . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) . The different Rag mutants used in the transfection are indicated at the top of each lane . ( c ) Rag regulates TORC1 but not TORC2 activity . Each indicated Rag construct ( 200 ng ) was co - transfected with either HA - S6K ( 20 ng ) , Myc - 4EBP1 ( 20 ng ) or of GST - Akt ( 100 ng ) . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

figf2(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane.(c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA-S6K (20 ng), Myc-4EBP1 (20 ng) or of GST-Akt (100 ng). Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.