Figure 2 ( A ) Representative confocal images of eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old WT mice sacrificed 3h after light on at 10 AM ( diurnal condition - LIGHT ) and 3h after light off at 10 PM ( nocturnal condition - DARK ) , nuclei were counterstained with DAPI ( blue ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions . ( B ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated on the X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . Mann and Whitney test * p ≤ 0 . 05 ( n = 3 mice for each time point ) . Values from 12h dark - adapted mice ( T0 ) were set to one . ( C ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old miR - 211 - / - and WT control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions . ( D ) qRT - PCR assay for Ezrin from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after light on at 10 AM . The graph shows the expression level of Ezrin normalized to Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) , * * * p ≤ 0 . 005 ( n = 6 mice ) . ( E ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR - 211 - / - mice . The graph shows the quantification of Ezrin normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 5 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * * p ≤ 0 . 01 . ( F ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old miR - 211 - / - mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one . ( G ) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one . ( H ) qRT - PCR assay for Mtmr10 and Klf13 from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after on at 10 AM . The graph shows the expression level of these genes normalized to the Hprt . Bar graphs represent mean values ± s . e . m . ( n = 3 mice )

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

Figure 2(A) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.(B) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated on the X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. Mann and Whitney test *p ≤ 0.05 (n=3 mice for each time point). Values from 12h dark-adapted mice (T0) were set to one.(C) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old miR-211-/- and WT control mice sacrificed 3h after light on at 10 AM (diurnal condition). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.(D) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).(E) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR-211-/- mice. The graph shows the quantification of Ezrin normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=5 mice) Mann and Whitney test (miR-211-/- vs WT), **p ≤ 0.01.(F) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old miR-211-/- mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one. (G) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one. (H) qRT-PCR assay for Mtmr10 and Klf13 from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after on at 10 AM. The graph shows the expression level of these genes normalized to the Hprt. Bar graphs represent mean values ± s.e.m. (n=3 mice)


Figure 3 ( A ) Representative images from ARPE - 19 cells treated with DMSO and NSC668394 . All cells were fixed and stained with anti - LC3 ( green ) and anti - LAMP1 ( red ) antibodies and nuclei were counterstained with DAPI ( blue ) . Scale bar 5 μm . ( B ) The plot shows the quantification of the numbers of LC3 / LAMP1 - positive vesicles per cell ( n ≥ 100 ) . Values represent means ± s . e . m from 3 independent experiments . Student ' s t - test ( NSC668394 vs DMSO ) * * * p ≤ 0 . 005 . ( C ) Representative Western blots of LAMP1 , EZRIN , EZRIN - pT567 , CLN5 , TRPML1 , SQSTM1 / p62 , CTSD and LC3 proteins from DMSO or NSC668394 treated cells . Note an increase of both proCTSD and its maturation CTSD heavy chain ( hc ) . The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control . Bar graphs represent mean values ± s . e . m . of at least n = 6 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 , * * * p ≤ 0 . 005 . ( D ) Representative images of RFP - GFP - LC3 assay in ARPE - 19 cells transiently transfected with RFP - GFP - LC3 and treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI ( blue ) . Scale bars : 5 μm . ( E ) Box plots showing quantitative analysis of RFP + GFP + puncta ( Autophagosome ) and RFP + GFP - puncta ( Autolysosome ) ( n ≥ 100 cells ) from 3 independent experiments . Box limits represent 25th percentile and 75th percentile ; horizontal lines represent medians ; whiskers display min . to max . values . Student ' s t - test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 . ( F ) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B ( CTSB ) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis . Scale bar 10 μm . ( G ) Graph showing the mean values of intensity of cresyl violet fluorescence ± s . e . m . of representative data ( n ≥ 100 cells ) from 3 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 . ( H ) Cathepsin B ( CTSB ) activity in lysates from NSC668394 - treated and DMSO - control ARPE - 19 cells . Graph showing the percentage of values ± s . e . m . of CTSB activity from 4 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

Figure 3(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005. (C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


Figure 4 ( A ) Representative images from HC assay of HeLaTFEB - GFP cells transfected with control ( siCTRL ) or siPPP3CB , and subjected to the indicated conditions . Analysis of TFEB nuclear translocation in HeLaTFEB - GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394 , silenced for EZRIN ( siEZR ) or serum - starved ( stv ) . Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB - GFP cells . Starvation ( stv ) was used as control . Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB . Scale bar 5 μm . ( B ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared to control ; n = 3 independent experiments were performed . Mann and Whitney test ( NSC668394 and stv vs DMSO ; siEZR vs siCTRL ) * p ≤ 0 . 05 . ( C ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells and subjected to the silencing of PPP3CB . At least 3 independent experiments were performed . Mann and Whitney test ( siPPP3CB vs siCTRL ) * p ≤ 0 . 05 . ( D ) qRT - PCR assay for PPP3CB from HeLaTFEB - GFP cells transfected with siPPP3CB and siCTRL . The graph shows the reduction in PPP3CB expression level normalized to the Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( siPPP3CB vs siCTRL ) , * * * p ≤ 0 . 005 ( n = 6 independent experiments ) . ( E ) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis . Torin treatment was used as control . ( F ) Representative traces of ML1 - GCaMP3 normalized fluorescence recorded in transiently transfected ARPE - 19 cells . During time - lapse recording , cells were stimulated with ML - SA1 ( 20 µM ) after addition ( arrowhead ) of DMSO or NSC668394 . Where indicated , the specific ML1 inhibitor ML - SI3 ( 10 µM ) was added . Bar graph reports the mean values ± s . e . m . of the time required by fluorescence to decay to half of the ML - SA1 - induced peak . n = 50 cells from 3 independent experiments . * * * p < 0 . 005 by Mann and Whitney test . ( G ) Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is reduced after Ca2 + chelator BAPTA treatment . Scale bar 5 μm . H ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared with DMSO under Ca2 + chelator BAPTA treatment . At least 3 independent experiments were performed . Mann and Whitney test ( stv , stv + BAPTA , BAPTA , NSC668394 , NSC668394 + BAPTA vs DMSO ) * p ≤ 0 . 05 . ( I ) qRT - PCR analysis for TFEB target genes ( TRPML1 , BECLIN , MAPLC3B and LAMP1 ) was performed on ARPE - 19 cells treated with DMSO or NSC668394 . Bar graphs represent mean values ± s . e . m . of at least 3 independent experiments . Mann and Whitney * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

Figure 4(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.(C) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells and subjected to the silencing of PPP3CB. At least 3 independent experiments were performed. Mann and Whitney test (siPPP3CB vs siCTRL) *p ≤ 0.05.(D) qRT-PCR assay for PPP3CB from HeLaTFEB-GFP cells transfected with siPPP3CB and siCTRL. The graph shows the reduction in PPP3CB expression level normalized to the Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (siPPP3CB vs siCTRL), ***p ≤ 0.005 (n=6 independent experiments).(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.


Figure 5 ( A ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) / anti - ZO - 1 ( red ) , anti - Ezrin - pT567 ( green ) / anti - ZO - 1 ( red ) , anti - Lamp1 ( green ) and anti - Sqstm1 / p62 ( green ) antibodies from 6 - month - old miR - 211 - / - sacrificed five months after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer . ( B ) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6 - month - old miR - 211 - / - mice . Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1 , Ezrin , Ezrin - pT567 , Sqstm1 / p62 and LC3 . The plots show the quantification of the indicated proteins normalized to the β - Actin loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) . Mann and Whitney test ( DMSO vs WT ; NSC668394 vs DMSO ) , * p ≤ 0 . 05 , * * * p ≤ 0 . 005 . ( C ) Cathepsin B ( CtsB ) activity in RPE lysates from miR - 211 - / - and WT mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . RPE was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . CtsB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) , * p ≤ 0 . 05 . ( D ) GUSB activity from retina from miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Retina was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . GUSB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of GUSB activity ± s . e . m . of independent experiments ( n = 3 mice ) . Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) .

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 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 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

Figure 5(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).


Figure 6 ( A ) Representative images of conventional TEM analysis of RPE of WT , DMSO - treated miR - 211 - / - mice and NSC668394 - treated miR - 211 - / - three - month - old mice . NSC668394 - treated miR - 211 - / - mice show rescue of accumulation of double - membrane phagolysosome - like structures containing poorly processed POS ( red arrows ) compared to DMSO - treated miR - 211 - / - mice . Scale bar 1 mm . Data information : ( OS ) outer segment ( RPE ) Retinal Pigment Epithelium . ( B ) Confocal images of representative eye cryosections immunostained with anti - Rhodopsin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment , which are sacrificed 3h after light on at 10 AM ( diurnal condition ) . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . The enlarged box highlights rhodopsin accumulation in the RPE ( green spots ) . Scale bar 5 μm . Autofluorescence from lipofuscin granules from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Representative images of retina cryosections immunostained with anti - Cone Arrestin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . N = at least 6 mice per group . Scale bar 100 μm . Data information : ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( RPE ) Retinal Pigment Epithelium . ( C ) Graphs showing number of lipofuscin granules from the RPE of mice as in ( B ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - DMSO vs WT and miR - 211 - / - NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 . * p ≤ 0 . 05 ( n = 6 mice ) . ( D ) Graphs show cone percentage ( cones / area ) from the retina of mice treated as in ( B ) . Error bars represent s . e . m . Mann and Whitney test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 ( n = 6 mice ) . ( E ) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394 - treated miR - 211 - / - mice ( green lines ) compared to DMSO - treated miR - 211 - / - control mice ( red lines ) . WT mice were used as a control ( black lines ) . Flicker recordings were performed with light intensities ranging from 10 − 4 to 15 cd s / m2 in steps of 0 . 6 logarithmic units at 6 Hz frequency . ( F ) Flicker responses , plotted as a function of stimulus intensity , from WT ( black lines ) , DMSO - treated miR - 211 - / - ( red lines ) and NSC668394 - treated miR - 211 - / - ( green lines ) mice , at three months of age . The amplitude of the recordings from NSC668394 miR - 211 - / - treated mice is significantly rescued compared to DMSO - treated miR - 211 - / - mice . WT mice were used as a control . Error bars represent s . e . m . ANOVA test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium.(B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.


Figure 1 . A representative example of a 7 days ( 7d ) old adult female posterior midgut at low ( A ) and high magnification ( B , Region 4 - Region 5 ) showing the initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) . Flies were reared under experimental regime that protects the intestinal epithelial integrity ( intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food , see Figure EV1 ) at 25 Co . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . The left and right images in all examples correspond to independent intestines of the same genotype and treatment . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 ) B . A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell - lineage tool . esg ( green cytoplasmic signal ) labels intestinal progenitor cells , whilst Histone - RFP ( red nuclear signal ) acts as a semi - permanent marker that allows the visualization of differentiated cells . Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone - RFP labelling , as an indication of negligible epithelial turnover . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . White dotted line outlines the gut using DAPI staining ( not shown ) as a reference . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 C . Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification ( GFP and Histone - RFP ) and different time points post ReDDM activation ( 3 - and 7 - days ) in experimental conditions that protect the epithelial integrity ; note that none of the cell populations in the gut ( GFP ( n . s . no significant , P = 0 . 5267 ) or Histone - RFP ( n . s . , P = 0 . 2752 ) significantly increase in number overtime ( Quantifications were made using N ≥ 2 biological replicates , total number of guts analysed at 3 days n = 34 and at 7 days n = 45 ; Mann - Whitney , C ) . Error bars represent Standard Error of the Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 ) D . A representative example of an adult female posterior midgut at low ( A ) and high magnification ( B , Region 4 - Region 5 ) showing the initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) after 16 hours of paraquat treatment in Oxford food at 25 Co ; note the expansion of the labelling with DBS - S - QF to large intestinal cells ( ECs ) ( compare D with A ) . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . White dotted line outlines the gut using DAPI staining as a reference . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 ) E . ReDDM lineage - tracing in an adult intestine reared in Oxford Medium and paraquat ( 20 mM ) during 16 hours at 29 Co ; note the abundance of Histone - RFP cells without GFP signal , as an indication of epithelial damage and subsequent differentiation of progenitor cells . The image on the left was acquired at low magnification with the 10x objective , while the right panel was acquired using the 40x . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 ) F . Quantification of ReDDM labelling at high magnification after paraquat treatment ; note the statistically significant increase ( * * ; P = 0 . 0099 ) of Histone - RFP expressing cells without GFP signal ( Quantifications were made using N ≥ 2 biological replicates ; Unpaired two - tailed t test , 3d n = 9 , 7d n = 7 ) . Error bars represent Standard Error of the Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 ) H . Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg - gal4 ( green immunostaining with antibody against P35 ) at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - P35 ( BL5073 ) / + I . ReDDM lineage - tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg - Gal4 and experimental conditions that protect the epithelial integrity at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 ) J . ReDDM quantification corresponding to the intestines described in ( I ) ; no significant increase in either esg ( n . s . , P = 0 . 1352 ) or Histone - RFP ( n . s . , P = 0 . 9801 ) cell number is observed ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , 3d n = 12 , 7d n = 11 ) . Error bars represent Standard Error of the Mean . DAPI ( blue ) labels the nuclei in panels A , D and H . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 representative example of a 7 days (7d) old adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA). Flies were reared under experimental regime that protects the intestinal epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food, see Figure EV1) at 25 Co. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. The left and right images in all examples correspond to independent intestines of the same genotype and treatment. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131) B. A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell-lineage tool. esg (green cytoplasmic signal) labels intestinal progenitor cells, whilst Histone-RFP (red nuclear signal) acts as a semi-permanent marker that allows the visualization of differentiated cells. Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone-RFP labelling, as an indication of negligible epithelial turnover. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining (not shown) as a reference. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019 C. Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification (GFP and Histone-RFP) and different time points post ReDDM activation (3- and 7-days) in experimental conditions that protect the epithelial integrity; note that none of the cell populations in the gut (GFP (n.s. no significant , P = 0.5267) or Histone-RFP (n.s. , P =0.2752) significantly increase in number overtime (Quantifications were made using N ≥ 2 biological replicates, total number of guts analysed at 3 days n =34 and at 7 days n=45; Mann-Whitney, C). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) D. A representative example of an adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA) after 16 hours of paraquat treatment in Oxford food at 25 Co; note the expansion of the labelling with DBS-S-QF to large intestinal cells (ECs) (compare D with A). The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining as a reference. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131)  E. ReDDM lineage-tracing in an adult intestine reared in Oxford Medium and paraquat (20 mM) during 16 hours at 29 Co; note the abundance of Histone-RFP cells without GFP signal, as an indication of epithelial damage and subsequent differentiation of progenitor cells. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) F. Quantification of ReDDM labelling at high magnification after paraquat treatment; note the statistically significant increase (**; P = 0.0099) of Histone-RFP expressing cells without GFP signal (Quantifications were made using N ≥ 2 biological replicates; Unpaired two-tailed t test, 3d n = 9, 7d n =7). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)  H. Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-gal4 (green immunostaining with antibody against P35) at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-P35 (BL5072); UAS-P35 (BL5073)/+ I. ReDDM lineage-tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-Gal4 and experimental conditions that protect the epithelial integrity at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073) J. ReDDM quantification corresponding to the intestines described in (I); no significant increase in either esg (n.s. , P = 0.1352) or Histone-RFP (n.s. , P = 0.9801) cell number is observed (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, 3d n = 12, 7d n = 11). Error bars represent Standard Error of the Mean. DAPI (blue) labels the nuclei in panels A, D and H. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073)


Figure 2A . ReDDM activation in Dronc heterozygous ( + / - ) intestine 7 days after temperature shift at 29 Co ; esg expression ( green ) labels the intestinal progenitor cells , Histone - RFP ( red ) is a semi - permanent marker retained in differentiated cells and Pdm - 1 ( grey ) labels differentiated ECs . The red arrows indicates the enlarged areas depicted in the insets in the entire figure . DAPI labels the DNA In the entire Figure ( blue ) . All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity ( intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food ) . B . ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc ( see full genotype in methods ) ; note the increased cell size of esg - expressing cells ( GFP + , green ) , the co - expression of Histone - RFP ( red ) and the EC marker Pdm - 1 ( gray ) , and the coexpression of Pdm - 1 and GFP in enlarged cells ( compare panels and detailed insets from A with B ) . The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - CherryC . Relative number of esg - expressing cells normalised to DAPI ; notice that the relative percentage of esg - labelled cells is significantly higher in Dronc fully mutant conditions ( - / - ) at 3 days ( * * * ; P = 0 . 0007 ) and 7d ( * * * * ; p < 0 . 0001 ) post temperature shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t - test , + / - n = 32 , - / - n = 25 ) . D . Average cell size of esg - expressing cells ( μm² ) ; note the increased cell size of Dronc fully mutant progenitor cells ( - / - ) at 3d ( * * ; P = 0 . 0014 ) and 7d ( * * * , P = 0 . 0007 ) post temperature shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed parametric t - test , 3d + / - n = 19 , 3d - / - n = 28 , 7d + / - n = 29 , 7d - / - n = 28 ) . E . Relative number of esg - negative cells expressing Histone - RFP normalised to DAPI ; notice that the number of Histone - RFP cells without esg expression is significantly higher in Dronc fully mutant ( - / - ) conditions at 7d ( * ; P = 0 . 0046 ) ( Quantifications were made using N biological replicates ≥ 2 ; Mann - Whitney test , + / - n = 24 , - / - n = 20 .

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 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 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 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

Figure 2A. ReDDM activation in Dronc heterozygous (+/-) intestine 7 days after temperature shift at 29 Co; esg expression (green) labels the intestinal progenitor cells, Histone-RFP (red) is a semi-permanent marker retained in differentiated cells and Pdm-1 (grey) labels differentiated ECs. The red arrows indicates the enlarged areas depicted in the insets in the entire figure. DAPI labels the DNA In the entire Figure (blue). All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food).B. ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc (see full genotype in methods); note the increased cell size of esg-expressing cells (GFP+, green), the co-expression of Histone-RFP (red) and the EC marker Pdm-1 (gray), and the coexpression of Pdm-1 and GFP in enlarged cells (compare panels and detailed insets from A with B). The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-CherryC. Relative number of esg-expressing cells normalised to DAPI; notice that the relative percentage of esg-labelled cells is significantly higher in Dronc fully mutant conditions (-/-) at 3 days (***; P = 0.0007) and 7d (****; p <0.0001) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t-test, +/- n=32, -/- n=25). D. Average cell size of esg-expressing cells (μm²); note the increased cell size of Dronc fully mutant progenitor cells (-/-) at 3d (**; P = 0.0014) and 7d (***, P = 0.0007) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed parametric t-test, 3d +/- n = 19, 3d -/- n = 28, 7d +/- n = 29, 7d -/- n = 28). E. Relative number of esg-negative cells expressing Histone-RFP normalised to DAPI; notice that the number of Histone-RFP cells without esg expression is significantly higher in Dronc fully mutant (-/-) conditions at 7d (*; P = 0.0046) (Quantifications were made using N biological replicates ≥ 2; Mann-Whitney test, +/- n = 24, -/- n = 20.


Figure 3 . A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal stem cells express GFP under the regulation of Dl - Gal4 . All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity ( flies transferred every two days into vials with fresh food ) . DAPI ( blue ) labels the DNA in the entire Figure . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / + ( recombinant chromosome made for this study ; the parental line delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP was obtained from Maria Dominguez ) B . A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under the regulation of Dl - Gal4 . Dl - expressing cells are labelled with UASGFP ( green ) ; there are no morphological differences between A and B . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Relative number of GFP + Delta - expressing cells normalised to DAPI ; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions ( ns ; P = 0 . 9231 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 22 , - / - n = 13 ) . Error bars represent Standard Deviation of the Mean . Genotypes : + / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry - / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry D . Average Delta cell size ( μm² ) ; notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs ( ns ; P = 0 . 9694 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 42 , - / - n = 21 ) . Error bars represent Standard Deviation of the mean . Genotypes : . A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal stem cells express GFP under the regulation of Su ( H ) - Gal4 . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under the regulation of Su ( H ) - Gal4 . Su ( H ) - Gal4 - expressing cells are labelled with UASGFP ( green ) and UAS - Histone - RFP ; note the increase in cell size when compared to E and the absence of Histone - RFP cells without GFP signal . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry Relative number of Su ( H ) - Gal4 - expressing cells normalised to DAPI ; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions ( n . s . ; P = 0 . 5099 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 30 , - / - n = 29 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry H . Average Su ( H ) cell size ( μm² ) , there is a significant increase in Su ( H ) Dronc null cells ( - / - ) when compared to the heterozygous control condition ( * * ; P = 0 . 0064 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 49 , - / - n = 33 ) . Error bars represent Standard Deviation of the mean . Genotypes :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 . A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes: +/- :w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry -/- : w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry D. Average Delta cell size (μm²); notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs (ns; P = 0.9694) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 42, -/- n = 21). Error bars represent Standard Deviation of the mean. Genotypes:. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Su(H)-Gal4. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Su(H)-Gal4. Su(H)-Gal4-expressing cells are labelled with UASGFP (green) and UAS-Histone-RFP; note the increase in cell size when compared to E and the absence of Histone-RFP cells without GFP signal. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry Relative number of Su(H)-Gal4-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (n.s. ; P = 0.5099) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 30, -/- n = 29). Error bars represent Standard Deviation of the mean. Genotypes: +/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ -/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry H. Average Su(H) cell size (μm²), there is a significant increase in Su(H) Dronc null cells (-/-) when compared to the heterozygous control condition (**; P = 0.0064) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 49, -/- n = 33). Error bars represent Standard Deviation of the mean. Genotypes:


Figure 4A . Representative image of a Drosophila posterior midgut showing the expression of Armadillo ( red membranes ) , Prospero ( red nuclei of EEs ) and Dronc - TurboID ( green shows Streptavidin labelled proteins by Dronc - TurboID ) ; note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells ( white arrows indicate an intestinal progenitor cell upregulating Dronc ) . Representative image of a Drosophila posterior midgut showing the expression of Armadillo ( red membranes ) and Actin - Dronc - GFP - Myc ( green , anti - GFP ) ; note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells ( arrow ) . High magnification of the picture shown in B . The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP + EBs . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc . Transversal Z - section of the cells shown in C . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc . E . The enteroblast marker Su ( H ) ( red , immunostaining against Beta - galactosidase ) strongly co - localises with high levels of Dronc expression ( green , immunostaining against GFP ) ; white arrow indicates the enlarged area depicted in the insets . F . There is extensive overlap between the expression of the EB marker Su ( H ) ( green , immunostaining against Beta - galactosidase ; white arrow ) and the apical caspase reporter DBS - S - QF ( red , immunostaining against HA ) , but it is also present in progenitor cells without Su ( H ) expression ( yellow arrowheads ) . DAPI ( Blue ) labels cell nuclei in all the panels .

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

Figure 4A. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes), Prospero (red nuclei of EEs) and Dronc-TurboID (green shows Streptavidin labelled proteins by Dronc-TurboID); note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells (white arrows indicate an intestinal progenitor cell upregulating Dronc).Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. Transversal Z-section of the cells shown in C. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets.F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.


Figure 5A . Representative ReDDM labeling of a Drosophila Dronc heterozygous ( + / - ) intestine overexpressing an RNAi against Notch for 3 days ; note the lack of fully differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) . DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + B . Representative ReDDM labelling of a Drosophila Dronc mutant homozygous ( - / - ) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC ; note the lack of differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) , as well as the increase of GFP - positive cells ( compare A to B ) . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Quantification of the number of Histone - RFP cells normalised to DAPI ( proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels ) ; note the statically significant increase in Histone - RFP positive cells in Dronc homozygous mutant intestines compared to controls ( * * ; P = 0 . 0080 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 8 , - / - n = 15 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 UAS - Notch - RNAi ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry D . Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC ; intestinal esg - positive progenitor cells ( green ( GFP ) and red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / + E . Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC ; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation ( compare D and E ) . The white arrows indicate the position of insets 500μm from the posterior region . Note the complete loss of esg labelled cells in this region . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry F . Relative number of esg - positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch - Intra ; note the significant reduction of esg - expressing cells ( * * * * ; P < 0 . 0001 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 17 , - / - n = 17 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - CherryG . Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC . esg expression in green ( GFP ) labels all intestinal progenitors cells , whilst Su ( H ) - lacZ ( red ) distinguishes a subpopulation of EBs within the esg - expressing cells . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + H . Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC . Note , the transcription of Su ( H ) in large cells GFP ( - ) , suggestive of an aberrant upregulation of Notch - signalling within fully differentiated ECs . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - CherryI . Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat . Note the absence of large Su ( H ) positive , GFP ( - ) cells compared to ( H ) . The white arrows indicate the enlarged area depicted in the insets . Genotypes : + / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry  D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / + E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118; esg-Gal4 UAS-CD8 -GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-CherryG. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-CherryI. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


figf1 ( a - c ) At 12 - 16 h post - transfection with GFP - LC3 , NIH3T3 ( a , b ) , MEF ( b ) or HCT116 ( c ) cells containing vector or the FLIP gene were treated with Hank ' s solution for 2 - 4 h ( a ) or with 2 μM rapamycin for 1 - 3 h ( b , c ) . Subsequently , GFP - LC3 was detected using an inverted fluorescence microscope . Scale bar , 10 μm ( a ) . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 ) ( b , c ) . At 60 h post - transfection with control siRNA or cFLIP siRNA , MEF cells were transfected with GFP - LC3 ( c ) . ( d ) Autophagosomes were visualized through scanning electron microscopy . Scale bar , 50 nm . The arrows indicate autophagosomes . Immunoblots ( IB ) of cFLIP and actin expression are shown in Supplementary Information , Fig . S1c . ( e , f ) NIH3T3 vector and NIH3T3 - KSHV - vFLIP cells ( e ) or TREX - BCBL - Vector and TREX - BCBL - vFLIP cells ( f ) were treated with Hank ' s solution or 2 μM rapamycin for 2 - 4 h , respectively , followed by IB with anti - LC3 and anti - actin antibody . ( g ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL - vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 2 μM rapamycin for an additional 12 h . GFP - LC3 was detected using an inverted fluorescence microscope . Scale bar , 5μm . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P 0 . 01 ) . ( h ) Autophagosomes were visualized by scanning electron microscopy . Scale bar , 50 nm .

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

figf1(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c).(d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c.(e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody.(g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01).(h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.


figf2 ( a ) BCBL cells were used for immunoprecipitation ( IP ) with mouse IgG or anti - Atg3 , followed by immunoblotting ( IB ) with anti - cFLIP antibody ( left panel ) . NIH3T3 - KSHV - vFLIP cells were used for IP with mouse IgG or anti - Flag antibody , followed by IB with anti - Atg3 antibody ( right panel ) . ( b ) FLIP and Atg3 interaction after autophagy induction . HCT116 cells were treated with or without 2 μM rapamycin or Hank ' s solution ( HBSS ) for 4 h before IP with anti - Atg3 antibody , followed by IB with anti - cFLIP antibody . ( c ) Schematic diagram of human Atg3 ( top ) . N , amino terminus ; FR , flexible region ; C , carboxy terminus . At 48 h post - transfection with GST or GST - Atg3 along with Flag - vFLIP ( left ) or GST or GST - Atg3 along with GFP - LC3 ( right ) , HEK293T cells were used for GST pulldown , followed by IB with the indicated antibodies . ( d ) Schematic diagram of KSHV vFLIP ( top ) . The black boxes indicate the K - α2 and K - α4 peptides . At 48 h post - transfection with V5 - Atg3 and either GST - vFLIP DED1 mutants ( middle ) or GST - vFLIP DED2 mutants ( bottom ) , HEK293T cells were used for GST pulldown , followed by IB with anti - V5 antibody . ( e ) At 48 h post - transfection with GST , GST - Atg3 , and either Flag - vFLIP or Flag - vFLIP mAtg3 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag antibody . ( f ) At 48 h post - transfection with GST - Atg3 and GFP - LC3 along with increasing amount of Flag - vFLIP , HEK293T cells were used for GST pulldown , followed by IB with anti - GFP or anti - Flag antibody . ( g ) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti - Atg3 antibody , followed by IB with anti - LC3 antibody . Finally , whole - cell lysates ( WCLs ) were used for IB with the indicated antibodies to show expression ( a - g ) .

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

figf2(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel).(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.(c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies.(d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody.(e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody.(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).


figf3 ( a ) TREX - BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy . The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis , autophagic death , apoptosis / autophagic death , and others forms of cell death . ( b ) At 12 - 16 h post - transfection with GFP - LC3 , doxycycline - treated TREX - BCBL vector , TREX - BCBL - vFLIP and TREX - BCBL - vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days ( top ) or 2 μM rapamycin for 12 h ( bottom ) . The percentage of cell death ( trypan blue staining ) and apoptosis level ( annexin V staining ) of rapamycin - treated cells was quantified as mean ± s . d . of three independent experiments . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope . Data are mean ± s . e . m . ; n = 200 - 300 cells ; three independent experiments ; * P < 0 . 01 . ( c ) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days . The percentage of cell death was determined by trypan blue staining . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 05 . ( d ) TREX - BCBL vector and TREX - BCBL - vFLIP cells were mock - treated or treated with 50 nM rapamycin for the indicated periods of time . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 001 . ( e ) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days . The results were quantified as mean ± s . d . of the combined results from three independent experiments ; * P 0 . 01 . Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer ( BC Z2 CS analyser ) were used to determine cell death and cell growth analysis ( b - e ) .

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

figf3(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death.(b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01.(c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05.(d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001.(e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).


figf4 ( b ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h . Trypan blue staining was then used to determine cell death ( as a percentage ) . Data are mean ± s . e . m . ; n = 200 - 300 ; three independent experiments ; * P 0 . 01 ; * * P 0 . 05 . ( c ) At 12 - 16 h post - transfection with GFP - LC3 , TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 30 μM of the TAT , K - α2 or K - α4 peptide ( top left ) or the TAT , C - α2 , or C - α4 peptide ( bottom left ) for an additional 12 h . Subsequently , autophagy was quantified as means ± s . d . of the combined results from three independent experiments ( 2 μM rapamycin treatment was included as a control ) . TREX - BCBL vector and TREX - BCBL - vFLIP cells were treated with doxycycline for 24 h , followed by incubation with 0 , 30 , or 50 μM of the TAT , K - α2 , or K - α4 peptide ( top right ) or the TAT , C - α2 , or C - α4 peptide ( bottom right ) for an additional 12 h and GFP - LC3 puncta were subsequently detected using an inverted fluorescence microscope . Scale bar , 5 μm . ( d ) KSHV - infected BCBL1 cells were treated with 30 μM of the K - α2 or the K - α4 peptide for 12 h and subjected to scanning electron microscopy . Scale bar , 50 nm . ( e ) TREX - BCBL - Vector and TREX - BCBL - vFLIP cells were treated with the K - α2 and the K - α4 ( 30 μM each ) peptide for 12 h , followed by immunoblotting ( IB ) with anti - LC3 and anti - actin antibody . ( f ) NOD / SCID mice received an injection of 5 × 106 BCBL1 - Luc cells , followed by intraperitoneal injections with 300 μg the TAT , K - α2 or Kα - 4 peptide for three weeks ( top three panels ) . After three weeks , a group of three mice treated with the TAT peptide was challenged with 300 μg of the K - α2 or Kα - 4 peptide for an additional three weeks ( bottom panel , see Supplementary Information , Fig . S15 ) . Tumours were measured by in vivo bioluminescence imaging .

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

figf4(b) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05.(c) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h and GFP-LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm.(d) KSHV-infected BCBL1 cells were treated with 30 μM of the K-α2 or the K-α4 peptide for 12 h and subjected to scanning electron microscopy. Scale bar, 50 nm.(e) TREX-BCBL-Vector and TREX-BCBL-vFLIP cells were treated with the K-α2 and the K-α4 (30 μM each) peptide for 12 h, followed by immunoblotting (IB) with anti-LC3 and anti-actin antibody.(f) NOD/SCID mice received an injection of 5 × 106 BCBL1-Luc cells, followed by intraperitoneal injections with 300 μg the TAT, K-α2 or Kα-4 peptide for three weeks (top three panels). After three weeks, a group of three mice treated with the TAT peptide was challenged with 300 μg of the K-α2 or Kα-4 peptide for an additional three weeks (bottom panel, see Supplementary Information, Fig. S15). Tumours were measured by in vivo bioluminescence imaging.