( e ) Ribosomes associated were immunopurified from starved wild - type ubp3Δ containing ( RPL25 - GFP ) ( 9 × MYC - UBI ) as indicated . GFP immunoprecipitates ( IP ; left ) total extracts ( TCA , right ) were analysed by immunoblotting with anti - Myc antibodies to reveal ubiquitinated ( top ) . Ub , ubiquitin . Bottom : immunoblotting with anti - GFP antibodies controls for presence of Rpl25p - GFP , anti - Rpl1p antibodies confirm equal loading of extract . asterisk marks G on beads recognized by secondary antibody .

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(e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25-GFP) and (9 × MYC-UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p-GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.


( a ) Structure of GBA1 gene on chromosome 1 schematic overview of gene correction using ZFNs . Black boxes represent exons . ( b ) Mutated gene - corrected ( GC ) iPSCs after sequencing ( upper lanes : iPSCs with heterozygous mutations ; lower lanes : iPSCs with homozygous wt - GBA1 sequences as indicated by arrows ) .

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(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).


Immunostaining of indicated differentiated iPSC cultures at DIV65 . were stained for TH ( red ) β - TubIII , FOXA2 , NURR1 , GIRK2 VMAT2 ( green ) . nuclei were counterstained with Hoechst ( blue ) . ( Bars , 20 μm ) .

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Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).


a ) Gating strategy used for neuronal sorting . Live as determined by FSC / SSC gating were selected for CD184 - APC - negative / low surface expression subsequently separated for CD24 - PE versus CD15 / CD29 / CD44 expression , detected in FITC channel as indicated in dot plots . Post - sort reanalysis shows representative samples of both fractions immediately after FACS enrichment , which were then plated for comparative in vitro ( phase contrast . Bars , 50 μm ) .

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a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 μm).


( b , c ) Immunocytochemistry at DIV2 post - sort for TH ( green ) neuronal marker MAP2 ( red ) performed on neuronal ( b ) mixed ( c ) populations of a representative control GBA - PD . Nuclei were counterstained with Hoechst ( blue ) . FACS enriches for both TH neuronal marker positivity in neuronal population compared with non - neuronal population . ( Bars , 50 μm . )

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(b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line. Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 μm.)


( a ) Ganglioside composition of human fibroblasts , iPSCs , differentiated iPSCs enriched iPSC - derived neurons ( after FACS ) from control ( C1 - 1 ) , GBA - PD ( PD1 - 1 ) GD ( GD1 - 1 ) subjects . lipids were metabolically labelled with [ 1 - 3H ] sphingosine visualized by digital autoradiography .

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(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography.


( b ) Activities of glycohydrolases ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) in control iPSCs iPSC - derived enriched neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - .

(b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test.


c ) GBA1 , GBA2 , β - galactosidase HEXA HEXB gene expression in control iPSCs iPSC - derived enriched neurons at DIV65 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 , Student ' s t - .

c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student's t-test.


( d ) Representative western blots showing GCase , GBA2 , β - galactosidase HEXA HEXB in control iPSCs corresponding iPSC - derived neurons ( DIV65 ) .

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(d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).


( a ) LC - MS / MS showing level of Glc - Cer in control , GBA - PD , isogenic controls GD iPSC - derived neurons . Data are represented as mean + s . d . ; n = 3 , * P0 . 01 , One - way ANOVA .

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(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA.


( b ) Control iPSCs ( C1 - 1 ) were differentiated for 65 days exposed to GCase inhibitor CBE ( 25 μM ) for 48 h . Representative western blot showing increased of α - syn in CBE - treated iPSC - derived neurons compared to untreated neurons .

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(b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons.


( c , d ) Representative western blots showing of α - syn in control , GBA - PD , gene - corrected isogenic controls GD iPSC - derived neurons .

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(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.


( e ) Optical densities of α - syn bands normalized by averaged value of GAPDH expressed as a of control ( CTRL ) value . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , one - way ANOVA . ( f ) Average α - syn normalized by averaged value of GAPDH expressed as a of respective isogenic mutant lines . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , Student ' s t - .

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(e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student's t-test.


( a ) Glycohydrolase enzyme activities ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) were measured in enriched neurons from control , GBA - PD , isogenic gene - corrected control GD iPSC - derived neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 compared with controls , one - way ANOVA .

(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA.


( b , c ) Representative western blots of GCase in control , GBA - PD , corresponding gene - corrected GD iPSC - derived neurons . Data are represented as mean + s . d . ; experiments were independently repeated three times . * P0 . 05 ; * * P0 . 01 , one - way ANOVA Student ' s t - .

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(b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.


CSF glycohydrolase activities were measured in with idiopathic PD ( iPD ) , GBA - PD - matched unaffected individuals ( CTRL ) . Enzyme activities were normalized to total concentrations expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . e . m . ; CTRL , n = 14 ; iPD , n = 15 ; GBA - PD , n = 15 . Experiments were performed in triplicate . * P0 . 05 ; * * P0 . 01 , Mann - Whitney U .

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CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann-Whitney U test.


( a ) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions . were stained for TH ( green ) , β - TubIII ( magenta ) LAMP1 ( red ) . nuclei were counterstained with Hoechst ( blue ) . High magnification insets are shown in right lane . ( Bars , 20 μm 10 μm in insets . )

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(a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), β-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnification insets are shown in the right lane. (Bars, 20 μm and 10 μm in the insets.)


( b ) Average number of LAMP1 + particles in TH + neurons were quantified by ImageJ . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , * * P0 . 001 , one - way ANOVA .

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(b) Average size and number of LAMP1+ particles in TH+ neurons were quantified by ImageJ. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, **P0.001, one-way ANOVA.


( c ) Western blot for LC3 in iPSC - derived neuronal cultures at DIV65 , untreated ( − ) or treated with 200 μM leupeptin 20 mM NH4Cl for 4 h ( + ) .

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(c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+).


( d ) of basal of LC3 - II relative to β - actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times . * P0 . 01 ; * * P0 . 001 , one - way ANOVA .

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(d) Quantification of the basal levels of LC3-II relative to β-actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times. *P0.01; **P0.001, one-way ANOVA.


( e ) of LC3 flux normalized to actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , one - way ANOVA .

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(e) Quantification of LC3 flux normalized to actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, one-way ANOVA.


( a ) calcium were evaluated in iPSC - derived neurons at basal conditions by measuring 340 / 380 nm of Fura - 2 acetoxymethyl ester . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - . ( Number of analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) . ( b ) Caffeine ( 10 mM ) was applied to iPSC - derived neurons , peak calcium was measured as a change in Fura - 2 . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , Student ' s t - . ( Number of analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) .

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(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62).


( c ) iPSC - derived neurons were treated with A23187 ( 0 - 5 μM ) for 4 h cytotoxicity was evaluated by released LDH activity . LDH release values were calculated as of untreated lysed by incubation with Triton X - 100 . Data are represented as mean + s . e . m . ; experiments were independently repeated four times in triplicate . * P0 . 05 , one - way ANOVA , control lines versus mutated lines ; δP0 . 05 , one - way ANOVA , mutated lines versus isogenic gene corrected controls .

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(c) iPSC-derived neurons were treated with A23187 (0-5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls.


( d ) of TH + treated with A23187 ( 1 μM ) for 4 h expressed as a of DMSO - treated controls . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 , one - way ANOVA .

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(d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA.


( e ) Knockdown efficiency of NECAB2 determined by qRT - PCR normalized to scrambled non - targeting shRNA . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * * * P0 . 0001 , Student ' s t - .

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(e) Knockdown efficiency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. ***P0.0001, Student's t-test.


( f ) Representative western blot for NECAB2 showing knockdown efficiency . 1 , scrambled non - targeting shRNA ; 2 , NECAB2 shRNA .

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(f) Representative western blot for NECAB2 showing knockdown efficiency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA.


( g ) of TH + in GBA - PD GD iPSC - derived neuronal cultures infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus treated with A23187 ( 1 μM for 4 h ) . Values were calculated as a of scrambled shRNA - treated . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , Student ' s t - .

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(g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student's t-test.


( h ) Representative showing GBA - PD iPSC - derived DA neurons infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus treated with A23187 ( 1 μM for 4 h ) . were stained for β - TubIII ( green ) TH ( red ) . Nuclei were counterstained with Hoechst ( blue ) . ( Bar , 20 μm . )

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(h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)


( B ) Serum CRAMP determination by ELISA ( n = 8 ) . Data information : Data in B were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

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(B) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) Localization expression of CRAMP ( red ) , E - cadherin ( green ) , F4 / 80 ( green ) Ly6G ( green ) in duodenum by immunofluorescent . Representative photomicrographs of merged were shown . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . Data information : Data were representative from three independent experiments .

(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.


( C ) Left , representative of duodenal damage by H & E . Scale bar : 200 μm . Right , graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

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(C) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Western blot densitometry analyses of duodenal tight junction ( claudin - 1 , occludin , ZO - 1 ZO - 2 ; n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( E ) Western blot densitometry of duodenal zonulin ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

(E) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Left , representative of duodenal damage by H & E . Scale bar : 200 μm . Right , graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot densitometry analyses of duodenal tight junction ( claudin - 1 , occludin , ZO - 1 ZO - 2 ; n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( F ) Western blot densitometry of duodenal zonulin ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( A ) Western blot densitometry analyses of gliadin - triggered inflammatory signaling [ p - EGFR ( Tyr1068 ) , p - Akt , Akt , MyD88 , TRAF6 , p - p65 p65 ] in epithelial ex vivo ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E - G ) percentages of ( E ) Foxp3 + Treg , ( F ) T - bet + Th1 ( G ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative were median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Duodenal colonizing P . aeruginosa A . muciniphila as determined by RT - qPCR using - specific species gene primers ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

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(D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) production of LasB ( a protease of P . aeruginosa ) was examined in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

(E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( H ) Duodenal colonizing PA14 PAO1 as determined by RT - qPCR using P . aeruginosa - specific species gene primer ( n = 9 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple

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(H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons


( C ) of CRAMP in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Representative of intestinal damage by H & E . Scale bar : 200 μm . graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot densitometry analyses of tight junction ( claudin - 1 occludin ) zonulin in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( I ) percentages of three subsets of CD45 + CD11c + DCs ( n = 8 ) . ( J - L ) percentages of ( J ) Foxp3 + Treg , ( K ) T - bet + Th1 ( L ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative were median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


A . Representative photomicrographs of mouse liver sections following a two - thirds PH of Ki67 positive hepatocytes ( brown ) visualized by . are representative of 3 independent per time point .

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A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point.


B . Western blot detection of p - MST1 , MST1 , p - LATS1 , LATS1 from isolated from homogenised liver tissue at several time points . TBP was used as a loading control . Results of a single experiment with n = 3 per group . Each band is from an independent . were normalized to TBP mean value of of p - MST to total MST p - LATS to total LATS is plotted .

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B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted.


C . enriched fractions obtained from sham - operated livers liver 48 h post PH were analyzed by Western blot for YAP expression . Sp1 was used as a loading control . Image is representative of a single experiment with n = 3 per group .

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C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group.


D . Photomicrograph of YAP1 expression in sham regenerating liver 48 h post PH visualized by immunoflourescence microscopy . are representative of 3 independent per condition .

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D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy. Images are representative of 3 independent animals per condition.


E . Livers from sham 48 h following PH were digested to isolate hepatocyte population . extracts were analyzed by Western blot for YAP TAZ expression . β - actin was used as a loading control . Results of a single experiment with n = 2 per group . Each band is representative of an independent .

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E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal.


F . RT - PCR of isolated from mouse liver for Birc5 , FoxM1B cyclin B1 . Data are presented as log2 fold change were calculated using non - operated mouse liver tissue as a control . Unpaired , two - Student t - was used to determine significance between log2 values of livers 6 , 24 48 h post PH compared to sham operated controls . Log2 values are plotted on a linear scale as mean ± SD .

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F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.


A - C . Representative photomicrograph of young ( 6 - 8 ) aged ( > 12 months ) livers 40 h post PH stained with Ki67 visualized by H E for histology . Sections were scanned using Histech Pannoramic MIDI Scanner Ki67 positive were quantitated with Quant Center 2 . 0 software . A proliferation index ( p . i . ) was calculated on number of Ki67 positive hepatocytes to total hepatocytes livers were classified as ( + ) regenerating , p . i . 10 % or ( - ) non - regenerating , p . i . 10 % . D . Number of young ( 6 - 8 ) aged ( 12 months ) mice with ( - ) non - regenerating ( + ) regenerating or livers on p . i . , 40 h post PH . deemed non - surviving for ethical reasons were euthanized 24 hours post PH . Representative results of a single experiment with n = 9 aged n = 6 young per group .

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A-C. Representative photomicrograph of young (6-8 weeks) and aged (>12 months) livers 40 h post PH stained with Ki67 and visualized by IHC and H E for histology. Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2.0 software. A proliferation index (p.i.) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as (+) regenerating, p.i.10% or (-) non-regenerating, p.i.10%.D. Number of young (6-8 weeks) and aged (12 months) mice with (-) non-regenerating and (+) regenerating or livers based on p.i., 40 h post PH. Animals deemed non-surviving for ethical reasons were euthanized 24 hours post PH. Representative results of a single experiment with n=9 aged and n=6 young animals per group.


A . Western blot detection of p - MST1 , MST , p - YAP , YAP TAZ in control 40 h following PH in young aged mice . TBP was used as a loading control . Representative results of a single experiment with n = 6 independent . Each band is from an independent . Western blot ratios are graphically depicted . were normalized to TBP mean value of group is plotted . Unpaired , two - Student t - was used to calculate significance between 0h ( resected liver ) 40h following PH . Error bars represent mean ±

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A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SD


B . RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 , Cyclin A2 , Cyclin D1 p21 . RT - PCR data are presented as log2 fold change each value post PH was calculated for each mouse by comparison to its resected liver lobe . Circle , with red outline marks aged non - regenerating livers shown in Figure 2B . Each dot is representing an independent .

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B. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.


A . Eight old mice were injected with liposomes coupled to scrambled siRNA ( siScr ) or sequences targeting MST1 MST2 ( siMST ) . Each dot is representing an independent . % of Mst1 Mst2 mRNA remaining in liver was tested by RT - PCR , 1 , 3 6 days post injection calculated compared to control non - transfected livers . Unpaired , two - Student t - was used to calculate significance of mRNA remaining in comparison to non - transfected control livers at each time point .

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A. Eight weeks old mice were injected with liposomes coupled to scrambled siRNA (siScr) or sequences targeting MST1 MST2 (siMST). Each dot is representing an independent animal. The % of Mst1 Mst2 mRNA remaining in the liver was tested by RT-PCR, 1, 3 and 6 days post injection and calculated compared to control non-transfected livers. Unpaired, two-tailed Student t-test was used to calculate the significance of mRNA remaining in comparison to non-transfected control livers at each time point.


B . Western blot detection of MST1 , p - LATS1 β - actin 1 , 3 6 days post siRNA injection . A representative from each time point is shown .

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B. Western blot detection of MST1, p-LATS1 and β-actin 1, 3 and 6 days post siRNA injection. A representative animal from each time point is shown.


C . Western blot detection of YAP1 , p - YAP1 , H3 p - H3 , 3 days post siRNA injection . Two representative from each group are shown . samples were run on same blot . blot is split due to non - consecutive loading of samples .

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C. Western blot detection of YAP1, p-YAP1, H3 and p-H3, 3 days post siRNA injection. Two representative animals from each group are shown. All samples were run on the same blot. The blot is split due to non-consecutive loading of the samples.


D . Quantitative RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 , 1 , 3 6 days post injection . Log2 fold change was calculated using non - transfected mouse liver as control . Unpaired two Student t - was used to calculate significance of fold change of in comparison to a panel non - transfected control livers .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, 1, 3 and 6 days post injection. Log2 fold change was calculated using non-transfected mouse liver as control. Unpaired two tailed Student t-test was used to calculate the significance of fold change of in comparison to a panel non-transfected control livers.


E . HNF4α Ki67 were detected by positive for both were classified as proliferating hepatocytes . Ki67 positive hepatocytes are indicated with an asterisk Ki67 negative bile / biliary with an arrow in tissue 3 days post siRNA delivery . percentages of positive hepatocytes at day 3 day 6 - post injection are graphed . Two - Student t - was used to calculate significance of of positive hepatocytes in comparison to non - transfected control livers .

E. HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes. Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts/biliary cells with an arrow in tissue 3 days post siRNA delivery. The percentages of positive hepatocytes at day 3 and day 6-post injection are graphed. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes in comparison to non-transfected control livers.


A . Aged mice ( > 12 months ) were injected i . v . with siScr or siMST . Twenty - four h after siRNA injection a 70 % PH was performed remnant / regenerating liver tissue was harvested 40 h later . p . i . of livers was calculated by of Ki67 positive hepatocytes on of positive liver were classified ( - ) non regenerating or ( + ) regenerating . Non - surviving were sacrificed before 40 h end time point . " n " indicates number of per group . B . Ki67 immunostaining of regenerating aged mice livers , 40 h post PH . Sections were scanned using Histotech Pannoramic MIDI Scanner quantitated with Quant Center 2 . 0 software . p . i . values are provided on image .

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A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. "n" indicates the number of animals per group.B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image.


C . Representative photomicrograph of an aged liver treated with siMST stained with H & amp ; amp ; E by for CK19 Ki67 . Arrows are indicating regions with no signs of reaction or oval expansion .

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C. Representative photomicrograph of an aged liver treated with siMST and stained with H&amp;E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion.


D . Quantitative RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 at 0 40 h post PH . Log2 fold change was calculated using non - transfected / non - resected mouse liver as a control . Representative results of a single experiment with n = 6 per group are shown . Paired , two - Student t - was used to calculate significant change in signals between liver tissues at time of resection ( 0h ) compared to 40 h post PH .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH.


E . of Ki67 positive hepatocytes in aged aged + siMST 40 hours post resection . Representative results of a single experiment with n = 6 independent aged for group are shown . Two - Student t - was used to calculate significance of of positive hepatocytes of siMST treated compared to non - treated controls .

E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls.


F . Endpoint liver was taken of remnant lobe expressed as a to total . Representative results of 1experiment with n = 6 independent for group expect for control group were n = 6 are shown . Estimated liver - to - before PH in mice = 3 . 85 ± 0 . 05 ( SD ) . Unpaired , two - Student t - was used to calculate significance of each aged group in comparison to control young group .

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F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group.


G . Hepatocyte geometric diameter was calculated ( see material methods ) . Representative results of 1 experiment with n = 3 independent per group . Unpaired , two - Student t - was used to calculate significant change in hepatocyte in liver tissue with without siMST 40 h post PH .

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G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.


( B ) of SARS - CoV - 2 infection . After siRNA knockdown of each target , viral infection was quantified as mean log2 fold - change ( log2FC ) of of SARS - CoV - 2 + relative to mean of scrambled non - targeting siRNAs ( " SCRAMBLED " , green dots ; accordingly mean log2FC value of scrambled non - targeting siRNAs was normalized to zero ) . Predicted positive targets ( " TARGETING " , grey dots ) , predicted negative targets ( " NEG " , blue dots ) positive controls ( " POS " , red dots , including ACE2 TMPRSS2 ) are shown . Wilcoxon rank - sum P value comparing predicted positive targets to scrambled non - targeting siRNAs is given .

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(B) Quantification of SARS-CoV-2 infection. After the siRNA knockdown of each target, viral infection was quantified as mean log2 fold-change (log2FC) of the percentage of SARS-CoV-2+ cells relative to the mean of scrambled non-targeting siRNAs ("SCRAMBLED", green dots; accordingly the mean log2FC value of scrambled non-targeting siRNAs was normalized to zero). Predicted positive targets ("TARGETING", grey dots), predicted negative targets ("NEG", blue dots) and positive controls ("POS", red dots, including ACE2 and TMPRSS2) are all shown. Wilcoxon rank-sum test P value comparing the predicted positive targets to scrambled non-targeting siRNAs is given.


( C ) of number after each target knockdown , calculated as mean fraction of DAPI + relative to scrambled non - targeting siRNAs ( i . e . latter was normalized to 1 ) . Colors of dots P value are interpreted in same way as in ( B ) .

(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).


( D ) Representative from siRNA showing SARS - CoV - 2 infection ( green channel , top row ) , number ( blue channel , middle row ) merged ( bottom row ) . Results for scrambled non - targeting siRNA as negative control ( left column ) , knockdown of three predicted top metabolic targets ( PIKFYVE , SLC16A10 , PIP5K1C , middle columns ) , knockdown of positive control ( ACE2 , right column ) are shown . Scale bar = 10 μm .

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(D) Representative fluorescence images from the siRNA assay showing SARS-CoV-2 infection (green channel, top row), cell number (blue channel, middle row) and merged (bottom row). Results for scrambled non-targeting siRNA as negative control (left column), knockdown of three predicted top metabolic targets (PIKFYVE, SLC16A10, and PIP5K1C, middle columns), and knockdown of positive control (ACE2, right column) are shown. Scale bar=10 μm.


( A ) PCA plot of gene expression profiles for Vero E6 samples from experimental groups : control ( no or remdesivir treatment ) , ( SARS - CoV - 2 - infected ) , + remdesivir ( SARS - CoV - 2 - infected treated by remdesivir ) , remdesivir ( remdesivir treatment alone without ) . There are 3 replicates in each group .

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(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.


( C ) PCA plot of average metabolic flux profile computed using iMAT algorithm ( Shlomi et al . 2008 ; Methods ) representative of each of experimental groups , labels are same as ( A ) .

(C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A).


( B ) Representative leak - subtracted ClC - 3 , ClC - 4 ClC - 5 whole - currents measured with ( upper row ) or without ( lower row ) 5 mM ATP added to pipette solution . Currents were elicited every 10 s by voltage steps from 0 mV to + 140 mV , followed by a step to - 100 mV to maximize off - gating currents visible as sharp peaks at end of pulses . recording started shortly ( 10s - 30s ) after obtaining a whole - configuration . first last recorded currents in a series are depicted in green red , respectively . NMDG - solutions were used to characterize ClC - 4 .

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

(B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4.


( C ) Effects of various adenine nucleotides or their washout on ionic transport of ClC - 3 , ClC - 4 ClC - 5 . current amplitudes were measured at end of pulses from experiments as shown in ( B ) normalized to initial current amplitudes obtained after establishing whole - configuration . Some of experimental data points have been omitted for clarity . Monoexponential fits to data are depicted as red lines . Asterisks indicate statistical significances evaluated by two - sample t - of residual currents determined at end of each 10 - minute measurement ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ; n = 5 - 11 ) .

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(C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11).


( D ) , Left panel : residual currents determined at end of 10 - minute measurements as shown in ( C ) . ( D ) , Right panel : time constants ( τ ) of current rundown obtained by fitting monoexponential functions to measurements as depicted in ( C ) , ( n = 5 - 11 ) . Asterisks indicate statistical significances evaluated using two - sample t - ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ) .

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(D), Left panel: residual currents determined at the end of 10-minute measurements as shown in (C). (D), Right panel: time constants (τ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in (C), (n= 5-11). Asterisks indicate statistical significances evaluated using two-sample t-test analysis (*, p < 0.05; ***, p < 0.001, n.s., not significant).


( B ) Voltage dependence of WT ClC - 5 off - gating charge normalized to ionic current at + 165 mV in absence or presence of adenine nucleotides ( n = 7 - 12 ) . Error bars represent . Red lines depict Boltzmann fits to data ; fit parameters are provided in Appendix Table S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1.


( C ) Voltage dependence of WT ClC - 3 off - gating charge normalized to ionic current at + 165 mV in absence or presence of adenine nucleotides ( n = 4 - 5 , Error bars represent . ) . Red lines represent Boltzmann fits to

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

(C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data


( A ) Representative whole - recordings of expressing E211G ClC - 5 upon voltage pulses to + 140 mV from a - 60 - mV holding potential applied in 10 - s intervals starting shortly after obtaining whole - configuration with ( left ) or without ( right ) 5 mM ATP in pipette solution . Leak subtraction was not applied . Asymmetric [ Cl - ] was used ( 110 mM internal NaCl substituted by TrisSO4 ) to achieve a stable negative reversal potential of < - 50 mV allowing to recognize recordings with high unspecific leak current contamination .

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

(A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination.


( B ) Normalized time course of steady - state E211G ClC - 5 current amplitudes measured with ( n = 5 ) or without ( n = 4 ) internal ATP . Error bars show . Red represents a monoexponential fit to data with a time constant of 108 ± 8 s ( ) ; currents measured with ATP - free pipette solution declined to 57 . 9 ± 2 . 6 % ( ) of initial amplitude .

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

(B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.


( A ) Effects of different adenine nucleotides or their washout on ClC - 5 current amplitudes ( n = 4 - 7 ) . data were normalized to initial current amplitude determined shortly after establishing whole - configuration . Some of data points are omitted for clarity . For comparison , ADP data set from Figure 1 is also depicted . Red lines indicate monoexponential fits to averaged data .

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

(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data.


( B ) Averaged residual currents determined after 600 - s dialysis as shown in ( A ) , normalized to current at start of measurements ( n = 4 - 7 ) . Asterisks indicate statistically significant differences from a two - sample t - with p < 0 . 001 ; n . d . - not defined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined.


Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in ( A ) .

Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).


A - D Appearance of autophagic structures ( marked with GFP - Atg8a ) during wound closure in epidermis of control third instar larvae after epidermal knockdown of autophagy pathway components Atg1 , Atg5 , Atg6 , Atg7 or Atg12 . constructs are expressed in epidermis under control of A58 - Gal4 driver . ( A ) Time points from movies of wounded epidermis . wounds have closed by 2 h in cases . ( B ) Higher magnification of areas marked by magenta boxes at t = 120 min . ( C ) of appearance of GFP - Atg8a in imaged ( 10000 µm2 ) 3 control larvae , shown for each larva . ( D ) of number of GFP - Atg8a in different genetic conditions measured in an of 10000 µm2 at time of wound closure ; n = 4 - 10 larvae each genotype , for detail see Data .

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

A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.


Control of epidermal autophagy by TOR signalling . ( A ) Epidermis of third instar larvae expressing autophagosome marker GFP - Atg8a together with constructs for up - or down - regulating autophagy pathway in epidermis . Healthy epidermis contains few autophagosomes , but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes . ( B ) Higher magnification of areas marked by magenta boxes in ( A ) .

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

Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).


Control of epidermal autophagy by TOR signalling . ( C ) of Atg8a in an of 10000 µm2 , n = 6 - 8 larvae each genotype . We assumed unequal sample unequal variances calculations were performed . Values are presented as box plots . Box plot elements are : center , median ; box limits , upper lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers .

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

Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.


A - D loss in photobleaching ( FLIP ) to free GFP motility within epidermis . A small ( magenta circle ; 179 or 1098 µm2 ) was - illuminated for indicated times ( 3 or 20 min ) in control or Atg1 - expressing epidermis also expressing free GFP . ( A ) Snapshots before bleaching at 3 points of recovery . ( B ) Kymographs along broken in ( A ) during recovery . ( C , D ) of recovery after bleaching , shown separately for 3 20 min bleaching protocols .

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A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.


B - D Atg1 - induced syncytium formation is abolished when Atg5 function is downregulated ( compare to rows 2 3 in Fig . 5A , B ) ; same representation as shown in Fig . 5A - D .

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B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.


C , D Accumulation of neutral lipid in cytoplasm under conditions of uncontrolled autophagy . ( C ) Fixed epidermis from larvae expressing indicated constructs was stained with BodiPy 493 / 503 to mark lipid accumulations . ( D ) of number of lipid in an epidermal of 10000 µm2 in indicated conditions . A - D , n = 7 - 20 larvae each genotype .

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

C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.


( A ) Wild ‐ type rho0 harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to ‐ acid or nitrogen starvation ( ‐ N ) medium supplemented with indicated carbon sources . were analysed at indicated time points by whole extraction western blot using α ‐ GFP α ‐ Cdc11 antibodies . of ATG8 induction is shown in lower left panel . Total GFP signals ( GFP ‐ Atg8 free GFP ) were quantified normalized to Cdc11 signals . Normalized values at 0 h were set as one relative changes are shown after 6 h starvation . of autophagic flux is shown in lower right panel as of free GFP to total GFP signals ( GFP ‐ Atg8 free GFP ) after 6 h starvation . means s . d . of four ( n = 4 ) independent experiments are indicated .

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

(A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) microscopical . Wild ‐ type , rho0 , Δatg7 expressing prATG8 ‐ GFP ‐ ATG8 were grown as described in ( A ) exposed to ‐ acid ( left panel , galactose ) or nitrogen starvation ( right panel , glucose ( ‐ N ) ) for 6 h . Vacuoles were visualized by over night FM4 ‐ 64 ( 1 μM ) ( red ) . Arrowhead indicates a punctate GFP ‐ Atg8 structure . Transmission microscopy were superimposed to visualize boundaries . localization of GFP signal was analysed in at least 150 ( n ⩾ 150 ) for each condition . Scale bars represent 1 . 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.


Autophagic response in with compromised respiratory chain III , IV , or V activity during ‐ acid starvation . Wild ‐ type , rho0 , atg7 mutants that are selectively inhibited in biogenesis of respiratory chain III ( CIII : cbs1 ) , IV ( CIV : mss51 , pet111 , pet122 ) , or F1Fo ‐ ATP synthase ( V ; CV : atp10 ) were exposed to ‐ acidstarvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type were exposed to antimycin A ( AA ) or oligomycin ( O ) during ‐ acid starvation period . strains expressed prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) . Samples were analysed as described in Figure 1A . means s . d . of five ( n = 5 ) independent experiments are indicated .

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

Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.


( A , B ) Wild ‐ type , rho0 , Δatg7 were exposed to ‐ acid starvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type were exposed to antimycin A ( AA ) or oligomycin ( O ) during ‐ acid starvation period . ATP from total ( upper panels ) or isolated mitochondria ( lower panels ) were determined at indicated time points .

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(A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points.


( C ) Wild ‐ type Δatg7 were exposed to ‐ acid starvation medium supplemented with acetate or galactose for 3 h . Wild ‐ type were exposed to antimycin A ( AA ) , oligomycin ( O ) , or CCCP ( 50 μM ) during ‐ acid starvation period when indicated . were treated with mitochondrial potential ‐ dependent dye DiOC6 ( 3 ) examined by microscopy . Average pixel intensities for at least 10 mitochondrial tubules ( n = 10 ) in 5 representative were determined for each condition . Values for wild ‐ type mitochondria in presence of acetate were defined as 100 % relative values were calculated accordingly . means s . d . are shown .

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

(C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.


( A ) Wild ‐ type , rho0 , Δnpr2 , Δnpr2 rho0 expressing prATG8 ‐ GFP ‐ ATG8 ( upper panel ) or prATG8 ‐ GFP ( lower panel ) were exposed to ‐ acid starvation medium supplemented with galactose in absence or presence of rapamycin . Samples were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated .

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(A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Wild ‐ type , rho0 , Δnpr2 , Δatg7 expressing prNPR1 ‐ NPR1 ‐ HA were exposed to ‐ acid starvation medium supplemented with galactose in absence ( upper panels ) or presence ( lower panels ) of rapamycin . hyperphosphorylated ( Npr1 ‐ P ) dephosphorylated ( Npr1 ) forms of Npr1 are indicated . were analysed at indicated time points by whole extraction western blot using α ‐ HA α ‐ Cdc11 antibodies .

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(B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies.


( C ) Wild ‐ type , rho0 , Δnpr2 , Δatg7 were exposed to ‐ acid starvation medium supplemented with galactose . Additionally , wild ‐ type were treated with rapamycin during starvation ( left panel ) . Phosphorylated ( Atg13 ‐ P ) dephosphorylated ( Atg13 ) Atg13 was monitored at indicated time points by whole extraction western blot using α ‐ Atg13 α ‐ Cdc11 antibodies .

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(C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.


( A ) PKA ‐ dependent regulation of autophagic response under ‐ acid starvation . Wild ‐ type , rho0 , pka , ras2G19V ‐ expressing harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to ‐ acid starvation medium supplemented with galactose . PKA activity in pka was inhibited by addition of 1NM ‐ PP1 ( PP1 ; 1 μg / ml ) . Samples were analysed as described in Figure 1A ; autophagic flux was determined after 3 6 h .

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(A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h.


( B ) In vivo activity of PKA . Wild ‐ type , rho0 , pka , ras2G19V ‐ expressing harbouring 6xMYC ‐ cki12 − 200 ( S125 / 130A ) ( Cki1 ) were grown in galactose medium . When indicated , wild ‐ type were grown in galactose medium in presence of antimycin A ( AA ) or oligomycin ( O ) for 6 h . PKA ‐ dependent phosphorylation of Cki1 was analysed by whole extraction western blot using a α ‐ Myc antibody ( upper panels ) . of phosphorylated ( Cki1 ‐ P ) non ‐ phosphorylated ( Cki1 ) forms of Cki1 relative to wild ‐ type ( wt = 1 ) ( lower panels ) .

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(B)In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels).


( C ) PKA inhibition restores autophagic flux , but not ATG8 induction in presence of mitochondrial dysfunction . Wild ‐ type , rho0 , pka , pka rho0 expressing prATG8 ‐ GFP ‐ ATG8 were treated as described in ( A ) . Samples were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated in ( A - C ) .

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(C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild‐type, rho0, pka, and pka rho0 cells expressing prATG8‐GFP‐ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (A-C).


( A ) ATG8 induction is independent of Atg1 - Atg13 autophagic flux . Wild ‐ type , rho0 , Δatg1 , Δatg7 , Δatg9 , Δatg11 harbouring prATG8 ‐ GFP ‐ ATG8 were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated .

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(A)ATG8 induction is independent of the Atg1-Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Atg1 Atg13 recruitment to PAS depends on mitochondrial function . Wild ‐ type rho0 harbouring prATG8 ‐ GFP ‐ ATG8 prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed to ‐ acid starvation medium supplemented with galactose for 3 h . Wild ‐ type were treated with antimycin A ( AA 30 ′ ) after 2 . 5 h of starvation for 30 min or with oligomycin ( O ) for 3 h of starvation . Arrowheads indicate position of GFP ‐ Atg8 . Transmission microscopy were superimposed to visualize boundaries . Scale bar represents 1 . 5 μm .

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(B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm.


( C ) Steady ‐ state of Atg1 ‐ Atg13 ‐ mCherry during ‐ acid starvation . Wild ‐ type , rho0 , Δatg7 expressing prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed treated as described in ( B ) analysed by whole extraction western blot using α ‐ dsRed α ‐ Cdc11 antibodies .

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(C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.


C , Growth curves for E . coli 113 / 3 in defined media in presence or absence of B12 ( o , r espectively ) . When challenged with 40 nM isolated R - domain ( ) , growth was largely impaired whereas growth in presence of 40 nM ColE9 W39A ( ▫ ) a pproached that of no colicin control , consistent with OmpF threading causing disengagement of ColE9 from BtuB . Error bars represent standard deviation across three biological replicates .

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C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, respectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) approached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.


ColE9 * denotes ColE9 H551A , an active site mutation that inactivates toxin . A , Widefield microscopy of E . coli JM83 labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment with or without prior treatment with CCCP . Each panel shows same in DIC ( grey ) , Hoechst stain ( blue ) ColE9 * K469CAF647 ( red ) . Overlays of Hoechst ColE9 * K469CAF647 are also shown . ColE9 * K469CAF647 remains bound to OM in presence of CCCP but this signal is lost on treatment with trypsin . Trypsin treatment in absence of CCCP yields some - associated ColE9 * K469CAF647 that likely represents internalised molecules ( see text ) . Scale bar , 1 µm .

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ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.


C , Box whisker plots showing mean pixel intensities for ColE9 * K469CAF647 per measured for indicated condition used , whiskers represent minimum maximum mean pixel , box shows 1st 3rd quartile with median shown as a . From left - to - right : E . coli JM83 ; E . coli btuB deletion showing loss of ColE9 * K469CAF647 - associated ; E . coli JM83 in presence of CCCP ; E . coli JM83 following trypsin treatment showing significant ColE9 * K469CAF647 remains associated with indicative of import ; E . coli JM83 treated with CCCP trypsin showing complete loss of internalised ColE9 * K469CAF647 . n , number of used , typically from 2 - 4 biological replicates . * * * * , indicates a t value < 0 . 05 in a student T - as a statistically significant result , ns indicates no significant difference as determined by student T - .

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C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.


B , Overnight plate assays comparing cytotoxic activity of wild - type ColE9 with ColE9 K469C labelled with AF488 , AF568 or AF647 against a lawn of E . coli with either OmpF ( JW2203 ) or OmpC ( JW0912 ) in outer ( shown as + OmpF + OmpC , respectively ) . Each plate was spotted with a serial dilution of colicin ( 10 µM - 2 . 4 pM , 4 - fold dilution per spot ) . Wild - type ColE9 was more active against E . coli with OmpF in outer than OmpC . Labelling ColE9 K469C with fluorophores reduced colicin activity in with OmpF in outer but largely abolished colicin activity in with OmpC .

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B, Overnight plate assays comparing the cytotoxic activity of wild-type ColE9 with ColE9 K469C labelled with AF488, AF568 or AF647 against a lawn of E. coli with either OmpF (JW2203) or OmpC (JW0912) in the outer membrane (shown as +OmpF and +OmpC, respectively). Each plate was spotted with a serial dilution of the colicin (10 µM - 2.4 pM, 4-fold dilution per spot). Wild-type ColE9 was more active against E. coli cells with OmpF in the outer membrane than OmpC. Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC.


C , Widefield microscopy of E . coli JW2203 ( + OmpF ) JW0912 ( + OmpC ) labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment . Each panel shows same in DIC ( grey ) , Hoechst stain ( blue ) of ColE9 * K469CAF647 ( red ) . Overlays of Hoechst ColE9 * K469CAF647 are also shown . Data show that following trypsin - treatment , significant ColE9 * K469CAF647 remains associated with ompF - expressing E . coli whereas little or no remains associated with ompC - expressing E . coli . Scale bar , 1 µm . D , Box whisker plots of E . coli JW2203 E . coli JW0912 labelled with ColE9 * K469CAF647 with without trypsin treatment : whiskers represent minimum maximum mean pixel , box shows 1st 3rd quartile with median shown as a . Microscopy data were collected as in c . mean pixel of ColE9 * K469CAF647 per was measured for each condition . n , number of , typically from 3 or 4 biological replicates . Data show that ColE9 * K469CAF647 translocates across OM through OmpF that this is impeded when E . coli has OmpC in outer .

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C, Widefield fluorescence microscopy images of E. coli JW2203 (+OmpF) and JW0912 (+OmpC) cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst stain (20 µM) for 30 min at 37 °C with or without trypsin treatment. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and fluorescence of ColE9* K469CAF647 (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. Data show that following trypsin-treatment, significant ColE9* K469CAF647 fluorescence remains associated with ompF-expressing E. coli whereas little or no fluorescence remains associated with ompC-expressing E. coli. Scale bar, 1 µm. D, Box and whisker plots of E. coli JW2203 and E. coli JW0912 cells labelled with ColE9* K469CAF647 with and without trypsin treatment: whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. Microscopy data were collected as in c. The mean pixel intensity of ColE9* K469CAF647 per cell was measured for each cell condition. n, number of cells, typically from 3 or 4 biological replicates. Data show that ColE9* K469CAF647 translocates across the OM through OmpF and that this is impeded when E. coli has OmpC in the outer membrane.


C . TIRF microscopy revealed time courses of CAR - GFP microcluster formation as CAR T spread on CD19 ICAM - 1 - coated SLBs .

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C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs.