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

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


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

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


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

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


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

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


Confocal 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 . of LAMP2 pixel on GSC # 9 transfected with mock FLAG - QKI . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 15 .

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


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

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


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

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


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

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


Fraction of surviving over time in GSCs # 1 # 9 , transduced with empty vector ( mock ) or FLAG - QKI bi - cistronic GFP plasmids . Data are plotted as of GFP - positive at day of ( Dx ) , normalized to starting point ( Day 4 post - infection , D4 ) . Data are representative of n = 3 .

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Fraction of surviving cells over time in GSCs #1 and #9, transduced with empty vector (mock) or FLAG-QKI bi-cistronic GFP plasmids. Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4). Data are representative of n=3.


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

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


Confocal of mTOR ( green ) LAMP2 ( red ) in GSC # 9 transfected with sic or siQKI , treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . of mTOR colocalization score with LAMP2 is shown . 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 mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 10 .

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Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( J ) ISLR level increased with nodal metastasis in colorectal cancer . 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 . solid horizontal lines represent median , box limits indicate interquartile ranges whiskers indicate limit superior limit inferior respectively .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( B ) of changes in control ( n = 9 ) cKO ( n = 10 ) mice during development .

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


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

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


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

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


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

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


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

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


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

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


( H ) of of cyclin D1 + Ki67 + in ( F G ) . n = 4 .

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


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

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


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

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


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

(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 Mob1 in colon epithelium from control cKO mice two days after 5 - day DSS treatment . β - actin was used as a loading control . of pMst1 / 2 versus Mst1 pMob1 versus Mob1 were shown under corresponding bands .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


A ) Representative of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 7 months of TgN3R182C150 at 18 months of . Representative show brain arteries of TgN3R182C150 ( 7 18 months ) , sham NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an α - SMA antibody ( green ) . Scale bar = 20µm . B ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of . NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per vessel increases significantly in TgN3R182C150 mice at 18 months ( n = 3 ) of versus NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of . Statistical significance was assessed using a Brown - Forsythe Welch ANOVA followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel ) : 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 : 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 mean ( ) .

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


A ) Representative of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 3 , 7 18 months of . Representative show brain arteries capillaries of TgN3R182C150 , sham NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an anti - perlecan antibody ( green ) . Scale bar = 20µm . B ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 - ECD stained average per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries capillaries between non - vaccinated 3 months old TgN3R182C150 ( n = 3 ) 7 months old TgN3R182C150 ( n = 6 ) mice 18 months old TgN3R182C150 ( n = 3 ) . of NOTCH3 - ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 - ECD stained average per vessel revealed a significant decrease in NOTCH3 - ECD deposition in brain arteries capillaries between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) non - vaccinated TgN3R182C150 ( n = 6 ) mice . Statistical significance was assessed using a Brown - Forsythe Welch ANOVA followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel ) : 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 : 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 dashed thicker lines are median .

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

A) 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.


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

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

A) 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 TgN3R182C150 mice was significantly reduced in vaccinated ( n = 10 ) TgN3R182C150 mice vs sham ( n = 9 ) . Statistical was performed using unpaired student t with Welch ' s correction . ( Sham vs Vaccinated * P = 0 . 0196 ) . Error bars indicate standard error of mean ( ) .

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

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).


A ) Representative show microglia stained with anti - CD68 antibody ( red ) Iba1 antibody ( green ) . Scale bar = 20µm . B ) of CD68 - stained revealed a significant increase in % of microglia microglia between N3 EGF1 - 5 - immunized ( n = 6 ) , sham ( n = 4 ) non - vaccinated TgN3R182C150 ( n = 5 ) mice . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons . ( % microglia with CD68 : 7 m . o . vs . Sham ns P = 0 . 7393 ; Sham vs . Vaccinated * * P = 0 . 0087 . CD68 ( % of microglia ) : 7 m . o . vs . Sham ns P = 0 . 9131 ; Sham vs . Vaccinated * P = 0 . 0157 , ns = non - significant ) . Error bars indicate standard error of mean ( ) .

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

A) 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 of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 7 months of stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an antibody against microglia ( Iba1 , green ) . Scale bar = 20µm . D ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per microglia revealed no alterations between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) non - vaccinated TgN3R182C150 ( n = 6 ) mice at 7 months of . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons . ( % 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 ) : 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 : 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 mean ( ) .

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

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).


C - F Division pattern of seam at postembryonic stage in hermaphrodites . Seam V1 - V4 V6 divide symmetrically at L2 stage ( highlighted in red ) , thus doubling in number .

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

C-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 ( shown in brackets ) gaps in alae ( between arrows ) .

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(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 retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants .

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

(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 showing expression of hbl ‐ 1 : : gfp : : hbl ‐ 1 reporter in VNC in wild type ( K ) , atg ‐ 2 ( L ) epg ‐ 6 ( M ) L3 larvae .

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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 of HBL ‐ 1 : : GFP in 50 unit areas in VNC in indicated strains .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains.


O - Q Confocal showing expression of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) reporter in wild type ( O ) , atg ‐ 2 ( P ) atg ‐ 3 ( Q ) late L4 larvae .

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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 of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal in indicated strains .

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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 of hbl ‐ 1 : : gfp in VNC col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated . Black lines in ( N , R - T ) represent average of reporter in each . P values are determined by Student ' s two ‐ unpaired t ‐ 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 ; miRISC , miRNA ‐ induced silencing ; RNAi , ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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

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.


A - C Wild ‐ type atg ‐ 2 hermaphrodites have a single vulva ( arrow ) . ( B ) let ‐ 60 ( n1046gf ) mutants show a Muv phenotype . Black arrows indicate pseudovulvae .

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A-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 Muv phenotype in let ‐ 60 ( n1046 ) mutants . number of adult 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 ‐ χ2 .

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

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 do not express ASEL ‐ specific reporter lim ‐ 6pro : : GFP .

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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 ‐ χ2 .

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

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.


AIN ‐ 1 : : GFP is weakly diffusely expressed in cytoplasm during embryogenesis in wild ‐ type . ( A ) Nomarski image of shown in ( B ) . Scale bar , 10 μm for whole . C . elegans remain same during embryogenesis loss of autophagy activity has no effect on . Thus , scale bars for are only shown once in each figure . ( C ) AIN ‐ 1 : : GFP forms a large number of in epg ‐ 6 mutant . ( A - C ) are confocal .

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AIN‐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 are dramatically elevated in autophagy mutant compared with wild type .

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(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 are formed in sqst ‐ 1 mutant .

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(E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos.


( F ) AIN ‐ 1 : : GFP accumulates into numerous in epg ‐ 7 mutant .

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(F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos.


( G , H ) Number of AIN ‐ 1 : : GFP ( G ) ALG ‐ 2 : : GFP ( H ) per focal plane in indicated strains . Error bars indicate s . d . of four examined .

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(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 cytoplasm in wild ‐ type . ( I ) Nomarski image of shown in ( J ) .

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(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 in epg ‐ 6 mutant . ( L ) ALG ‐ 2 : : GFP forms a large number of in epg ‐ 6 mutant under stress conditions . GFP , green ; s . d . , standard deviation .

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(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.


( A - D ) Both AIN ‐ 1 : : GFP SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed diffusely localized in wild ‐ type .

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(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 colocalize with SQST ‐ 1 in atg ‐ 3 mutant . Scale bars , 10 μm for insets that show a magnified view .

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(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 are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant .

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(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 with LGG ‐ 1 in epg ‐ 6 mutant .

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(M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos.


( Q ) % of AIN ‐ 1 : : GFP colocalized with SQST ‐ 1 , SEPA ‐ 1 LGG ‐ 1 in autophagy mutants . Error bars indicate s . d . of four examined .

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(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 shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo .

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(R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo.


( S ) Direct interaction between AIN ‐ 1 LGG ‐ 1 in an in vitro pull ‐ down . 5 % of His fusion used for pull ‐ down are shown as input . Corresponding Ponceau ‐ stained blots show amount of used for pull ‐ down .

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(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 .

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(T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay.


( a ) Knockdown of dRagA dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 untreated ( lane 2 ) or treated with indicated RNAi were starved of for 1 h followed by acid stimulation for 30 min Phosphorylation of dS6K were determined by immunoblotting with indicated antibodies .

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(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 dRagC are not required for dAkt phosphorylation . Drosophila S2 untreated ( lanes 1 - 3 ) or treated with indicated RNAi were starved of for 1 h stimulated with for 30 min . Phosphorylation of dAkt were determined by immunoblotting with appropriate antibodies as indicated . NC , negative control RNAi .

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(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 dRagC function in parallel with TSC2 PTEN . dRagA / or dRagC RNAi was added to S2 in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) this increase was compromised by dRagA or / dRagC RNAi . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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(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.


( a ) Constitutively active RagA 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 . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation were determined by immunoblotting with appropriate antibodies , as indicated .

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(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 ) . different Rag mutants used in transfection are indicated at top of each lane .

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(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 were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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(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.


( a ) RagAQ66L RagCS75N activate TORC1 in absence of . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) into HEK293 . were starved of for 1 h before collection . Phosphorylation were determined by immunoblotting with appropriate antibodies , as indicated .

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(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( b ) RagAT21N RagCQ120L block S6K phosphorylation in response to acid ( AA ) stimulation . pcDNA3 ( 200 ng , lanes 1 2 ) or each indicated Rag construct was co - transfected with HA - S6K into HEK293 . were starved of for 1 h ( - AA ) either remained in starvation medium or were stimulated with - acid - containing - medium ( + AA ) for 30 min before collection . Phosphorylation were determined by immunoblotting with appropriate antibodies , as indicated .

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(b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were starved of amino acids for 1 h (-AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( c ) RagAT21N RagCQ120L suppress insulin - induced stimulation of S6K phosphorylation . pcDNA3 ( 200 ng , lanes 1 2 ) or each indicated Rag construct was co - transfected with HA - S6K ( 20 ng ) or GST - Akt ( 100 ng ) into HeLa . were serum - starved overnight stimulated with insulin ( 400 nM ) for 30 min . Phosphorylation were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. 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.


( a ) dRagA positively regulates wing compartment . Wild - type or mutant dRagA transgenes were expressed in posterior compartments with en - GAL4 driver . ratios of representative posterior to anterior compartment areas are shown . Posterior compartment was significantly increased in response to dRagAQ61L expression decreased in response to dRagAT61N expression . Data are mean ± s . d . , * P = 7 . 32 × 10 − 4 ( n = 7 ) , * * P = 6 . 18 × 10 − 4 ( n = 12 ) , Student ' s two - t - , ( n is number of adult wings analysed ) .

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(a) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed).


( b ) dRagA positively regulates wing . average of posterior compartment from en - GAL4 UAS - dRagA adult wings is shown . expressing dRagAQ61L are significantly larger dRagAT61N - expressing are smaller than controls . Data are mean ± s . d . , * P = 1 . 15 × 10 − 3 ( n = 7 ) , * * P = 0 . 025 ( n = 12 ) , Student ' s 2 - t - ( n represents number of adult wings analysed ) .

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(b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed).


( c , d ) dRag GTPases positively regulate larval fat . ( c ) of clonally - induced dRagA - expressing or dRagC homozygous mutant relative to neighbouring wild - type control is shown . was determined from phalloidin - stained fixed fat samples from fed or 48 h starved larvae . Expression of dRagAWT or dRagAQ61L significantly increased relative under starvation but not fed conditions . expressing dRagAT61N dRagC loss - of - function were significantly smaller than control only under nutrient replete conditions . Data are means ± s . d . , * P = 2 . 04 × 10 − 3 ( n = 5 ) , * * P = 2 . 94 × 10 − 6 ( n = 14 ) , * * * P = 3 . 79 × 10 − 7 ( n = 14 ) , * * * * P = 1 . 36 × 10 − 5 ( n = 30 ) , Student ' s two - t - . ( d ) Representative examples of fat with altered dRagA activity . expressing dRagA transgenes are marked by expression of GFP in left middle panels , dRagC homozygous mutant are marked by absence of GFP in right panel . Scale bar represents 50 μm .

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

(c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.


( a ) Rag acts through TORC1 to regulate S6K phosphorylation . HEK293 were transfected with constructs as indicated . Co - expression of of mTORKD construct ( 600 ng ) or rapamycin treatment ( rapa , 20 nM , 30 min ) abolished effect of RagAQ66L RagCS75N on S6K phosphorylation . level of mTORKD was determined by immunoblotting with anti - mTOR antibody .

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(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody.


( b ) RagA / RagC TSC1 / TSC2 independently regulate S6K phosphorylation . HEK293 were transfected with 200 ng of each Rag / or ′ constructs as indicated . acid starvation for 1 h ( - AA ) is indicated . Phosphorylation of transfected were determined by immunoblotting with appropriate antibodies , as indicated .

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(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.


( c ) TSC2 RagA / B independently affect S6K phosphorylation . HA - S6K ( 20 ng ) was transfected into HeLa with or without RNAi against human TSC2 , RagA RagB as indicated .

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(c) TSC2 and RagA/B independently affect S6K phosphorylation. HA-S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated.


( d ) RagAT21N RagCQ120L do not block Rheb - induced S6K phosphorylation . RagAT21N RagCQ120L ( 200 ng each ) were transfected into HEK293 with or without Rheb construct ( 20 ng ) . S6K was included in co - transfection . Phosphorylation of transfected were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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(d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a , b ) dRagC is not required for Rheb - induced growth . ( a ) of Rheb - overexpressing in control or dRagC mutant ( dRagC − / − ) backgrounds under fed conditions , relative to that of neighbouring control which were assigned a value of 1 . Overexpression of Rheb led to a significant increase in in both control dRagC mutant backgrounds . Data are mean ± s . d . , * P = 0 . 034 ( n = 5 ) , * * P = 6 . 1 × 10 − 3 ( n = 5 ) , Student ' s two - t - ( n represents number of experimental samples ) . ( b ) A representative example of a clone of Rheb - overexpressing in a dRagC − / − . Rheb transgene - expressing are marked by co - expression of GFP . boundaries are labelled by phalloidin in red ; nuclei are labelled by DAPI in blue . Scale bar represents 50 μm .

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

(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm.


( c , d ) Expression of dRagAQ61L fails to rescue growth impairment of Rheb mutant . ( c ) Relative of clonally - induced Rheb26 . 2 homozygous mutant in a control background in expressing dRagAQ61L throughout fat . Clonally induced Rheb26 . 2 homozygous mutant were significantly smaller than neighbouring control both in wild - type in dRagAQ61L expressing backgrounds . Data are mean ± s . d . , * P = 2 . 91 × 10 − 4 ( n = 7 ) , * * P = 2 . 59 × 10 − 8 ( n = 5 ) , Student ' s two - t - ( fed conditions where n represents number of experimental samples ) . ( d ) A representative example of Rheb homozygous mutant ( marked by lack of GFP , arrows ) in fat ubiquitously expressing UAS - dRagAQ61L . GFP - positive control in this experiment are a mixture of Rheb + / − Rheb + / + . Scale bar represents 50 μm .

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

(c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.


( a - d ) dRagAQ61L suppresses autophagy . ( a ) Drosophila fat clonally expressing dRagAQ61L ( marked in green by GFP expression ) failed to accumulate autolysosomes ( shown in surrounding control by punctate Lysotracker Red ) in response to starvation for 4 h . Nuclei are marked in blue by DAPI . ( b - d ) Induction of autophagosomes in response to 4 - h starvation is shown by punctate pattern of GFP - Atg8a expression in control fat ( b ) , but not in expressing dRagAQ61L ( c ) . average number of GFP - Atg8a - marked autophagosomes per in control dRagAQ61L - expressing clones is shown ( d ) . Data are mean ± s . d . , * P = 2 . 91 × 10 − 6 , Students two - t - ( n = 33 fat samples imaged per genotype ) . Scale bars represent 25 μm in each panel .

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

(a-d) dRagAQ61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagAQ61L (marked in green by GFP expression) failed to accumulate autolysosomes (shown in surrounding control cells by punctate Lysotracker Red staining) in response to starvation for 4 h. Nuclei are marked in blue by DAPI. (b-d) Induction of autophagosomes in response to 4-h starvation is shown by the punctate pattern of GFP-Atg8a expression in control fat body cells (b), but not in cells expressing dRagAQ61L (c). The average number of GFP-Atg8a-marked autophagosomes per cell in control and dRagAQ61L-expressing clones is shown (d). Data are mean ± s.d., *P = 2.91 × 10−6, Students two-tailed t-test (n = 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel.


( e ) RagA regulates LC3 conversion in mammalian . Myc - LC3 was co - transfected with RagAQL RagCSN or RagATN RagCQL into HEK293 as indicated . One day after transfection , were cultured in - acid - sufficienct medium ( + AA ) or - acid - depleted medium ( − AA ) for 4 h before collection . Western blotting for Myc - LC3 HA - Rag were performed . Autophagic conversion of LC3I into lipidated LC3II form was blocked by active RagA stimulated by dominant - negative RagA . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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

(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a , b ) dRagA activation increases sensitivity to starvation . Expression of dRagAQ61L using fat - - specific Cg - GAL4 driver significantly decreased survival of adult flies under starvation ( b ) but not fed ( a ) conditions , relative to controls ( Cg - GAL4 alone ) . Asterisks indicate significant difference , compared with controls . Data are mean ± s . d . , * P 0 . 05 , Students two - t - ( n = 150 flies / genotype / treatment ) .

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

(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagAQ61L using the fat-body-specific Cg-GAL4 driver significantly decreased survival of adult female flies under starvation (b) but not fed (a) conditions, relative to controls (Cg-GAL4 alone). Asterisks indicate significant difference, compared with controls. Data are mean ± s.d., *P 0.05, Students two-tailed t-test (n = 150 flies/genotype/treatment).


I complexes involving aggregators . Heatmaps show absolute difference in solubility change after heat shock between each member . complexes with at least 75 % of its members quantified containing at least 60 % of its members as aggregators are shown . ' repair ' = ' repair NEIL2 - PNK - Pol ( beta ) - LigIII ( alpha ) - XRCC1 ' .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O

I Protein complexes involving aggregators. Heatmaps show the absolute difference in solubility change after heat shock between each complex member. Protein complexes with at least 75% of its members quantified and containing at least 60% of its members as aggregators are shown. 'DNA repair complex' = 'DNA repair complex NEIL2-PNK-Pol(beta)-LigIII(alpha)-XRCC1'.