Luc - CTL assay was performed with MCF7 cells transfected with control or PD - L1 - specific siRNAs and cocultured with or without CTLs and bsAb . For each condition , the luciferase activity of PD - L1 - siRNA - treated cells was normalized to that of the control siRNA ; n = 8 . Error bars denote ± SEM .

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Luc-CTL assay was performed with MCF7 cells transfected with control or PD-L1-specific siRNAs and cocultured with or without CTLs and bsAb. For each condition, the luciferase activity of PD-L1-siRNA-treated cells was normalized to that of the control siRNA; n = 8. Error bars denote ± SEM.


Comparison between the Luc - CTL assay ( ▪ ) and the classical chromium ( Cr ) - release assay ( ○ ) with MCF7 as target cells and survivin - specific T cells as effector cells at varying effector to target ( E : T ) ratios . Error bars denote ± SEM .

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Comparison between the Luc-CTL assay (▪) and the classical chromium (Cr)-release assay (○) with MCF7 as target cells and survivin-specific T cells as effector cells at varying effector to target (E:T) ratios. Error bars denote ± SEM.


B Correlation between replicates for screen 2 for both viability and toxicity sets , as determined by the Pearson rank correlation test .

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B Correlation between replicates for screen 2 for both viability and toxicity sets, as determined by the Pearson rank correlation test.


C - E Graphical summary of gene function related to modification of T cell - mediated tumor lysis and cellviability for screens 1 , 2 , and 3 , respectively . Positive score = reduced cancer cellviability ; negative score = increased viability . X - axis : influence on cellviability without addition of T cells . Y - axis : influence on cellviability with addition of T cells . Appropriate immune - suppressive ( PD - L1 , CEACAM - 6 , GAL - 3 ) and lethality ( UBC , PLK - 1 ) controls , along with few positive ( GRM4 , GRK5 ) and negative ( CCR9 , IL8 ) candidate immune - modulatory hits are highlighted herein .

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C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.


A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT - PCR ( upper ) and immunoblot ( lower ) analysis , respectively . GAPDH and beta - actin were used as controls for RNA and protein normalization , respectively .

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A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT-PCR (upper) and immunoblot (lower) analysis, respectively. GAPDH and beta-actin were used as controls for RNA and protein normalization, respectively.


B Luc - CTL cytotoxicity assay with PBMC - derived CTLs and bi - specific Ab as effector population and MCF7 as target cells , which were transfected with individual ( s1 - s4 ) or pooled CCR9 siRNA sequences . PD - L1 and non - specific control siRNAs were used as positive and negative controls , respectively , for CTL - mediated cytotoxicity .

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B Luc-CTL cytotoxicity assay with PBMC-derived CTLs and bi-specific Ab as effector population and MCF7 as target cells, which were transfected with individual (s1-s4) or pooled CCR9 siRNA sequences. PD-L1 and non-specific control siRNAs were used as positive and negative controls, respectively, for CTL-mediated cytotoxicity.


C , D Cr - release assay showing % specific lysis of MCF7 cells by survivin - specific T cells at different ratios upon CCR9 knockdown ( C ) or overexpression ( D ) . MCF7 cells were transfected with either CCR9 siRNA s1 ( Δ ) , pooled siRNA sequences ( ○ ) , positive control PD - L1 ( □ ) , and non - specific control siRNA ( ▪ ) ( C ) or with pCMV6 - AC - His control vector ( ▪ ) and pCMV6 - AC - His - CCR9 expression construct ( ○ ) ( D ) 72 h prior to the assay .

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C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (Δ), pooled siRNA sequences (○), positive control PD-L1 (□), and non-specific control siRNA (▪) (C) or with pCMV6-AC-His control vector (▪) and pCMV6-AC-His-CCR9 expression construct (○) (D) 72 h prior to the assay.


E Cr - release assay showing % specific lysis of MDA - MB - 231 breast tumor cell line by survivin - specific T cells at different ratios upon CCR9 knockdown ( ○ ) in comparison to the control knockdown ( ▪ ) .

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E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown (○) in comparison to the control knockdown (▪).


F , G Cr - release assay showing lysis of patient - derived melanoma cells ( M579 - A2 ) by tumor - infiltrating lymphocytes ( TIL 412 ) ( F ) or lysis of PANC - 1 pancreatic adenocarcinoma cells by patient - derived pancreatic TIL 53 ( G ) at different E : T ratios upon CCR9 ( ○ ) or control ( ▪ ) knockdown .

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F, G Cr-release assay showing lysis of patient-derived melanoma cells (M579-A2) by tumor-infiltrating lymphocytes (TIL 412) (F) or lysis of PANC-1 pancreatic adenocarcinoma cells by patient-derived pancreatic TIL 53 (G) at different E:T ratios upon CCR9 (○) or control (▪) knockdown.


A , B ELISpot assay showing IFN - γ ( A ) and granzyme B ( B ) secretion by survivin - specific T cells , as spot numbers , upon CCR9 knockdown ( black bars ) in MCF7 cells compared to the control knockdown ( white bars ) . T cells ( TC ) alone ( grey bars ) were used as control for background spot numbers .

A, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.


C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin - specific TC and either CCR9hi MCF7 ( transfected with CCR9 - specific siRNA ) or CCR9lo MCF7 ( transfected with control siRNA ) cells .

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C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.


D Phospho - plex analysis showing the phospho - STAT levels in survivin - specific TC upon encountering CCR9hi or CCR9lo MCF7 cells . Log2 ratio of mean fluorescent intensity ( MFI ) of the respective analytes to the unstimulated TC is plotted herein .

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D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.


E Immunoblot analysis showing the phospho - STAT1 levels in the CCR9hi - treated , CCR9lo - treated or unstimulated TC using the phospho - specific STAT1 ( pTyr701 ) antibody . Beta - actin was used as the loading control .

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E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.


A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines . CCR9 knockdown ( k . d . ) in MCF7 cells was achieved using specific shRNA ( see Materials and Methods ) .

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A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods).


B Cr - release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 ( □ ) or CCR9 ( ○ ) inhibition using specific siRNAs in comparison to the control siRNA ( ▪ ) . Mean ± SEM are depicted herein .

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B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 (□) or CCR9 (○) inhibition using specific siRNAs in comparison to the control siRNA (▪). Mean ± SEM are depicted herein.


C MCF7 cells were transfected with control or CCR9 - specific siRNAs , and 48 h later , the supernatants ( CCR9lo or CCR9hi SSN , respectively ) were used to culture survivin TCs overnight . Supernatant - treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr - release assay along with wild - type MCF7 cells . Mean ± SEM are depicted herein .

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C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR9lo or CCR9hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. Mean ± SEM are depicted herein.


D Cr - release assay showing % specific lysis of MCF7 cells that were pre - treated with or without pertussis toxin ( PTX ) , or knocked down for CCR9 using specific siRNA . Mean ± SEM are depicted herein .

D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. Mean ± SEM are depicted herein.


E , F MCF7 cells transfected with control siRNA ( CCR9hi ) or CCR9 siRNA ( CCR9lo ) were cocultured with survivin TCs for 12 h . Gene microarray was performed with the total RNA extracted from purified T cells after the coculture . Volcano plot ( E ) illustrating fold change ( FC ; log2 ) in gene expression intensities compared with P - value ( − log2 ) between CCR9hi - and CCR9lo - treated TCs . Horizontal bar at y = 4 . 32 represents a statistical significance of P = 0 . 05 ( genes in gray below this line did not reach significance ) . LogFC cutoff at ± 0 . 5 is represented by the vertical lines . Heatmap representation of the top upregulated ( LogFC > 0 . 5 ) and downregulated ( LogFC < − 0 . 85 ) genes ( F ) with P ≤ 0 . 05 . Individual replicates per sample group are shown herein .

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E, F MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein.


A Cr - release assay showing TIL 209 - mediated lysis of CCR9 + M579 - A2 ( transduced with control shRNA ) or CCR9 − M579 - A2 cells ( transduced with CCR9 - specific shRNA ) . Curves represent mean ± SEM .

A Cr-release assay showing TIL 209-mediated lysis of CCR9+ M579-A2 (transduced with control shRNA) or CCR9− M579-A2 cells (transduced with CCR9-specific shRNA). Curves represent mean ± SEM.


C , D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9 + or CCR9 − M579 - A2tumors in TIL - treated mice ( C ) or the PBS alone group ( D ) . Statistical difference was calculated using the unpaired one - sided Mann - Whitney U - test .

C, D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9+ or CCR9−M579-A2tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


( A ) Confocal laser scanning micrographs showing the localization of a functional CDKA ; 1 : mVenus fusion protein in the wildtype ( WT ) and cartoons on top highlighting the changes in abundance of CDKA ; 1 : mVenus in the nucleus and cytoplasm during the course of meiosis . The region colored in beige represents the cytoplasm , in green the nucleoplasm , and in white the nucleolus . Scale bar : 10 μm .

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(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.


( B ) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA ; 1 : mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis . Error bars represent mean ± SD and two biological replicates were performed .

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(B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.


( C ) Immunolocalization of CDKA ; 1 ( green ) and ASY1 ( red ) on spread chromsomes in leptotene and zygotene of wild - type plants expressing a functional PROCDKA ; 1 : CDKA ; 1 : Strep construct . The last lane shows a magnification of the region marked by the red rectangle . Arrowheads indicate synapsed regions of homologous chromosomes where CDKA ; 1 is no longer present . Scale bar : 5 μm .

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(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.


( D ) Chromosome spread analysis of the wildtype and the hypomorphic cdka ; 1 mutant CDKA ; 1T161D . ( a , h ) zygotene or zygotene - like stages ; ( b , i ) pachytene or pachytene - like stages ; ( c , j , k ) diakinesis or dikinesis - like stages ; ( d ) metaphase I ; ( e , i , m , n ) end of meiosis I with two ( e , m ) or three ( i ) pools of chromosomes ; ( f ) metaphase II ; ( g ) tetrad . Red arrowheads indicate the initiated formation of a phragmoplast . White arrowheads depict mitochondria . Scale bars : 10 μm .

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(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.


( A ) ASY1 : GFP and ASY1T142D : GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA ; 1T161D mutants . ASY3 : RFP , highlighting chromsomes , was used as a marker for the staging of meiosis . Scale bar : 5 μm .

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(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.


( B ) Kinase assays of CDKA ; 1 - SDS , - TAM , and - CYCA3 ; 1 complexes using ASY1 puried from baculovirus - infected insect cells as a substrate . The upper panel shows the autoradiograph . The control reaction without CDKA ; 1 - cyclin complex indicates a background activity co - purified from insect cells . The lower panel indicates protein loading by coomassie briliant blue ( CBB ) staining . Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay . Numbers indicate the relative intensities of ASY1 bands .

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(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.


( C ) The upper panel shows a phos - tag gel analysis of ASY11 - 300 and ASY11 - 300 / T142V ; T184V with and without CDKA ; 1 - SDS kinase complexes using an anti - MBP antibody . The lower panel denotes loading of CDKA ; 1 using an anti - Strep antibody . Arrowheads represent the proteins as indicated .

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(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.


( B ) Localization patterns of different ASY1 : GFP variants together with ASY3 : RFP ( for staging of zygotene and pachytene ) in a asy1 mutant background during prophase I . Bar : 5

(B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ


( C ) Signal distribution profiles of ASY1 : GFP and ASY1T142V : GFP at leptotene The many small peaks with low amplitude in ASY1T142V : GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype .

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(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.


( D , E ) Immunolocalization of ASY1 ( D ) and ZYP1 ( E ) in ASY1 : GFP ( asy1 ) and ASY1T142V : GFP ( asy1 ) plants using anti - GFP and anti - ZYP1 antibodies , respectively . DNA was stained with DAPI ( blue ) . Scale bars : 5 μm .

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(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.


( A ) Yeast two - hybrid interaction assays of ASY3 with different ASY1 variants . Monomeric GFP ( mGFP ) fused with AD ( activating domain ) and BD ( binding domain ) were used as controls . Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp ( - L / T , left panel ) , on Synthetic Drop - out ( SD ) medium in the absence of Leu , Trp , and His ( - L / T / H , middle panel ) , and on SD medium in the absence of Leu , Trp , His and Ade ( - L / T / H / A , right panel ) . Yeast cells were incubated until OD600 = 1 and then diluted 10 - , 100 - and 1000 - fold for the assays .

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(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.


( B ) GST pull down of ASY3 with different ASY1 variants . The numbers above the bands show the relative intensity of the bands . The input and pull down fractions were analyzed by immuno - blotting with the anti - GST ( ASY3 ) and anti - MBP ( ASY1 ) antibodies . ( C ) Quantification of the pull down fractions of ASY1 as shown in ( B ) . The band intensity in the pull down of ASY11 - 300 / T142V ; T184V at a Triton X - 100 concentration of 0 . 5 % was defined as 1 . The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled - down ASY3 fraction . The average band intensity of ASY1 at different concentrations of Triton X - 100 used was plotted . Asteriks indicate significant difference ( two - tailed t - test , p < 0 . 05 ) . Error bars represent mean ± SD and two biological replicates were performed . .

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(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..


( A ) Localization patterns of ASY1T142V ; T184V : GFP together with ASY3 - RFP in the wildtype and pch2 mutants . Please note that images of ASY1T142V ; T184V : GFP in pch2 mutants were taken with increased sensitivity for a better visibility . Scale bar : 5 μm .

(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.


( B ) Seed sets ( mean ± SD , n = 5 ) of WT , asy1 , pch2 , asy1 pch2 , ASY1T142V ; T184V : GFP ( asy1 ) and ASY1T142V ; T184V : GFP ( asy1 pch2 ) plants . Asteriks indicate significant differences ( two - tailed t - test , P < 0 . 01 ) and ns depicts no significant difference . Scale bar : 2 mm .

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(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.


( C ) Localization patterns of ASY1 : GFP , ASY1T142V ; T184V : GFP , and ASY11 - 570 : GFP in the wildtype ( WT ) and / or in pch2 mutant plants at early prophase I . Scale bars : 5 μm .

(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.


( D ) Localization pattern of PCH2 : GFP together with ASY1 : RFP in the male meiocytes of wildtype . Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2 . Scale bar : 10 μm .

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(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.


( B ) Interaction assays of different ASY1 fragments ( with and without the closure motif ) .

(B) Interaction assays of different ASY1 fragments (with and without the closure motif).


( C ) Interaction analysis of the ASY1 closure motif ( ASY1571 - 596 ) with different ASY1 HORMA domain variants .

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(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.


PI ( 5 ) P Regulates Autophagosome Biogenesis ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells treated with carrier alone , or in combination with PI ( 5 ) P di - C16 at indicated concentrations for 1 hr , in the absence and presence of 400 nM BAF ( treated in combination with lipids ) . Note that LC3 - I is often very faint compared with LC3 - II in HeLa cells under the protein extraction conditions we used ( see Figure S1C ) . However , this was not a problem because one should relate LC3 - II to tubulin ( mean ± SEM ) .

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PI(5)P Regulates Autophagosome Biogenesis(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with carrier alone, or in combination with PI(5)P di-C16 at indicated concentrations for 1 hr, in the absence and presence of 400 nM BAF (treated in combination with lipids). Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we used (see Figure S1C). However, this was not a problem because one should relate LC3-II to tubulin (mean ± SEM).


PI ( 5 ) P Regulates Autophagosome Biogenesis ( C and D ) HeLa cells stably expressing GFP - LC3 were treated as in ( A ) and then left in complete media ( basal ) or starvation media ( HBSS ) for 2 hr , then fixed and analyzed on a Cellomics ArrayScan system . Quantification of numbers of GFP - LC3 vesicles per cell in the different conditions is shown in ( D ) ( mean ± SEM ) .

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PI(5)P Regulates Autophagosome Biogenesis(C and D) HeLa cells stably expressing GFP-LC3 were treated as in (A) and then left in complete media (basal) or starvation media (HBSS) for 2 hr, then fixed and analyzed on a Cellomics ArrayScan system. Quantification of numbers of GFP-LC3 vesicles per cell in the different conditions is shown in (D) (mean ± SEM).


( E ) HeLa cells stably expressing GFP - mRFP - LC3 were treated as in ( A ) and analyzed on a Cellomics ArrayScan system . Quantification of numbers of autophagic vesicles ( AV ) or autolysosomes ( AL ) per cell in the different conditions is shown in the graph ( mean ± SEM ) .

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(E) HeLa cells stably expressing GFP-mRFP-LC3 were treated as in (A) and analyzed on a Cellomics ArrayScan system. Quantification of numbers of autophagic vesicles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph (mean ± SEM).


( F and G ) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti - HA antibody in HeLa cells transfected with HA - ATG12 and ATG5 and loaded with exogenous lipids . We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5 - ATG12 conjugation efficiency ( mean ± SEM ) .

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(F and G) Western blot analysis of free ATG12 and ATG5ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and loaded with exogenous lipids. We quantified the ratio of ATG5ATG12 versus free ATG12 by direct infrared fluorescence detection on an Odyssey Infrared Imaging System to assess ATG5-ATG12 conjugation efficiency (mean ± SEM).


( H ) HeLa cells transfected with GFP - PHD3X or GFP - PHD3X Znmut and RFP - LC3 for 16 hr were left in complete media ( basal ) or starvation media ( HBSS ) for 1 hr , then fixed and imaged on a confocal microscope . Empty arrowheads indicate PHD3X single - labeled structures , while filled arrowheads indicate PHD3X - LC3 - and PHD3X - ATG16 - positive structures . Bar , 10 μm .

(H) HeLa cells transfected with GFP-PHD3X or GFP-PHD3X Znmut and RFP-LC3 for 16 hr were left in complete media (basal) or starvation media (HBSS) for 1 hr, then fixed and imaged on a confocal microscope. Empty arrowheads indicate PHD3X single-labeled structures, while filled arrowheads indicate PHD3X-LC3- and PHD3X-ATG16-positive structures. Bar, 10 μm.


( I ) HeLa cells transfected with GFP - PHD3X and RFP - LC3 for 16 hr were left in HBSS for 1 hr , then fixed and imaged on Elyra superresolution microscope . Final visualization was performed in Volocity 6 . 3 software using isosurface rendering of selected cropped regions of the data sets . Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface . Representative cropped regions from different cells are shown . Bar , 1 . 13 μm . See also Figure S1 .

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(I) HeLa cells transfected with GFP-PHD3X and RFP-LC3 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed in Volocity 6.3 software using isosurface rendering of selected cropped regions of the data sets. Note that this rendering means that vesicles positive for green and red do not look yellow but have green and red on the surface. Representative cropped regions from different cells are shown. Bar, 1.13 μm. See also Figure S1.


( B and C ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM - 201636 for 2 hr in the presence or absence of BAF ( mean ± SEM ) .

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(B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM).


( D and E ) HeLa cells transiently transfected with Strawberry - ATG16L1 were treated with YM - 201636 ( 100 nM , 2 hr ) in HBSS , then fixed and stained for endogenous WIPI - 2 . Bar , 10 μm . ( E ) Quantification of ATG16L1 and WIPI2 structures per cell , n = 20 cells ( mean ± SEM ; n = 3 independent experiments ; ∗ ∗ ∗ p < 0 . 001 , t test ) .

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(D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test).


( F ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells transfected with HA - ATG12 and ATG5 and treated with YM - 201636 ( 100 nM , 2 hr ) ( mean ± SEM ) .

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(F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM).


( G ) HeLa cells transfected with GFP - PHD3X and Strawberry - ATG16L1 for 16 hr were left in HBSS for 1 hr , then fixed and imaged on Elyra superresolution microscope . Final visualization was performed as previously described . Bar , 1 . 13 μm .

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(G) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in HBSS for 1 hr, then fixed and imaged on Elyra superresolution microscope. Final visualization was performed as previously described. Bar, 1.13 μm.


( H and I ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in cells incubated with YM - 201636 ( 100 nM , 2 hr ) and loaded with exogenous PI ( 5 ) P for the last 1 hr in the presence or absence of BAF ( mean ± SEM ) . See also Figure S2 .

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(H and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5)P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.


( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells transfected for 5 days with two rounds of control , PI5P4K2A , 2B , or 2C siRNA either left untreated or treated with BAF ( 200 nM , 16 hr ) ( mean ± SEM ) .

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(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).


( C ) HeLa cells stably expressing GFP - mRFP - LC3 treated as in ( A ) were analyzed on a Cellomics ArrayScan system as previously described ( mean ± SEM ) .

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(C) HeLa cells stably expressing GFP-mRFP-LC3 treated as in (A) were analyzed on a Cellomics ArrayScan system as previously described (mean ± SEM).


( D ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA and transfected with HA - ATG12 and ATG5 for the last 16 hr ( mean ± SEM ) .

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(D) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA and transfected with HA-ATG12 and ATG5 for the last 16 hr (mean ± SEM).


( E and F ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , or catalytic - dead PI5P4K2C along with RFP - LC3 for 16 hr ( E ) or 30 hr ( F ) were fixed and imaged on a confocal microscope . Bar , 10 μm . ( G ) Quantification of cells ( percentage of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in the different conditions from ( F ) is shown in the graph ; n = 200 cells ( mean ± SEM ) . See also Figure S3 .

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(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.


( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI ( 5 ) P di - C16 ( 10 μM ) in the absence and presence of BAF ( mean ± SEM ) .

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(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).


( C ) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV - PI5P4K2C overexpression construct for 48 hr were transfected with EGFP - httQ74 for the last 48 hr . Percentage of cells with EGFP - positive aggregates is shown in the graph ; n = 500 cells ( mean ± SEM ) . Cells expressing control vector typically have 25 % of aggregates and we set the control at 100 % to enable comparison and statistics from independent experiments .

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(C) HeLa cells treated with PI5P4K2A or PI5P4K2C siRNA for 5 days or with pCMV-PI5P4K2C overexpression construct for 48 hr were transfected with EGFP-httQ74 for the last 48 hr. Percentage of cells with EGFP-positive aggregates is shown in the graph; n = 500 cells (mean ± SEM). Cells expressing control vector typically have 25% of aggregates and we set the control at 100% to enable comparison and statistics from independent experiments.


( D and E ) Percentage of cells with EGFP - positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP - httQ74 for the last 48 hr ( D ) , or in COS7 and SKNSH cells ( E ) , transfected with PI5P4K2C plasmid together with EGFP - httQ74 for the last 48 hr .

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(D and E) Percentage of cells with EGFP-positive aggregates was scored in HEK293 cells treated with PI5P4K2C siRNA for 5 days and cotransfected with EGFP-httQ74 for the last 48 hr (D), or in COS7 and SKNSH cells (E), transfected with PI5P4K2C plasmid together with EGFP-httQ74 for the last 48 hr.


( F and G ) WT ( Atg5 + / + ) or autophagy - deficient cells lacking the key autophagy gene Atg5 ( Atg5 − / − ) MEFs were treated with PI5P4K2C siRNA ( 5 days ) or PI5P4K2C plasmid ( 48 hr ) together with EGFP - httQ74 for 48 hr . The percentages of transfected cells with EGFP - httQ74 aggregates were assessed and shown in the graph ; n = 500 cells ( mean ± SEM ) . Representative confocal images are shown in ( G ) . See also Figures S3 and S4 .

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(F and G) WT (Atg5+/+) or autophagy-deficient cells lacking the key autophagy gene Atg5 (Atg5−/−) MEFs were treated with PI5P4K2C siRNA (5 days) or PI5P4K2C plasmid (48 hr) together with EGFP-httQ74 for 48 hr. The percentages of transfected cells with EGFP-httQ74 aggregates were assessed and shown in the graph; n = 500 cells (mean ± SEM). Representative confocal images are shown in (G). See also Figures S3 and S4.


PI ( 5 ) P Triggers Autophagy in the Absence of VPS34 Activation ( A and B ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells pretreated with Wm ( 200 nM , 2 hr ) , then loaded with 10 μM PI ( 5 ) P for 1 hr ( in the presence of Wm and BAF ) ( mean ± SEM ) .

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PI(5)P Triggers Autophagy in the Absence of VPS34 Activation(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells pretreated with Wm (200 nM, 2 hr), then loaded with 10 μM PI(5)P for 1 hr (in the presence of Wm and BAF) (mean ± SEM).


( C ) Quantification of numbers of GFP - LC3 vesicles in HeLa cells stably expressing GFP - LC3 treated as in ( A ) and then shifted to starvation media ( HBSS ) or complete media ( Basal ) for 2 hr ( in the presence of Wm ) ( mean ± SEM ) .

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(C) Quantification of numbers of GFP-LC3 vesicles in HeLa cells stably expressing GFP-LC3 treated as in (A) and then shifted to starvation media (HBSS) or complete media (Basal) for 2 hr (in the presence of Wm) (mean ± SEM).


( D ) HeLa cells transfected with HA - ATG12 and ATG5 treated with 200 nM Wm as in ( A ) and subjected to western blot analysis with anti - HA antibody to detect free ATG12 and the ATG5 - ATG12 complex ( mean ± SEM ) . ATG5 - ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti - ATG12 antibody ( bottom ) .

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(D) HeLa cells transfected with HA-ATG12 and ATG5 treated with 200 nM Wm as in (A) and subjected to western blot analysis with anti-HA antibody to detect free ATG12 and the ATG5-ATG12 complex (mean ± SEM). ATG5-ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti-ATG12 antibody (bottom).


( E and F ) Western blot analysis of LC3 - II and tubulin levels and quantification of LC3 - II / tubulin ratio in HeLa cells transfected with control , PI5P4K2A , 2B , and 2C siRNA and treated with Wm ( 200 nM , 2 hr in the presence of BAF ) ( mean ± SEM ) .

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(E and F) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected with control, PI5P4K2A, 2B, and 2C siRNA and treated with Wm (200 nM, 2 hr in the presence of BAF) (mean ± SEM).


( G and H ) Western blot analysis of free ATG12 and ATG5 - ATG12 complex levels with anti - HA antibody in HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA , transfected with HA - ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr ( mean ± SEM ) .

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(G and H) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA, transfected with HA-ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr (mean ± SEM).


( I ) HeLa cells stably expressing GFP - LC3 treated with control , PI5P4K2A , 2B , and 2C siRNA , were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr ( in the presence of Wm ) ( mean ± SEM ) .

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(I) HeLa cells stably expressing GFP-LC3 treated with control, PI5P4K2A, 2B, and 2C siRNA, were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr (in the presence of Wm) (mean ± SEM).


( J and K ) HeLa cells stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , and myc - MTMR3C413S , incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm . Cells were fixed , stained with anti - myc antibodies , and imaged by confocal microscope . Asterisks indicate transfected cells . Quantification of numbers of GFP - LC3vesicles per cell is shown in ( K ) ( mean ± SEM ) .

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(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3vesicles per cell is shown in (K) (mean ± SEM).


( J and K ) HeLa cells stably expressing GFP - LC3 were transfected for 30 hr with myc - tagged empty vector , myc - MTMR3WT , and myc - MTMR3C413S , incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm . Cells were fixed , stained with anti - myc antibodies , and imaged by confocal microscope . Asterisks indicate transfected cells . Quantification of numbers of GFP - LC3 vesicles per cell is shown in ( K ) ( mean ± SEM ) .

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(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3 vesicles per cell is shown in (K) (mean ± SEM).


( L and M ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , and RFP - LC3 for 30 hr were loaded with 10 μM PI ( 3 ) P for 1 hr in complete medium , and then fixed and imaged on confocal microscope . Bar , 10 μm . Quantification of cells ( percentage of total ) showing more than 10 RFP - LC3 vesicles in the different conditions from ( J ) is shown in ( M ) ; n = 200 cells ( mean ± SEM ) . See also Figure S5 .

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(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3)P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.


( A ) HeLa cells transfected with GFP - WIPI2B or GFP - DFCP1 preloaded with indicated concentrations of PI ( 5 ) P for 1 hr , starved in HBSS for 1 hr , and then incubated with Wm in HBSS were tracked by time - lapse microscopy for 10 min after the addition of Wm . Quantification of WIPI2B or DFCP1vesicles ( percentage of those at the starting time [ T0 ] ) during the treatments are shown in the graphs .

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(A) HeLa cells transfected with GFP-WIPI2B or GFP-DFCP1 preloaded with indicated concentrations of PI(5)P for 1 hr, starved in HBSS for 1 hr, and then incubated with Wm in HBSS were tracked by time-lapse microscopy for 10 min after the addition of Wm. Quantification of WIPI2B or DFCP1vesicles (percentage of those at the starting time [T0]) during the treatments are shown in the graphs.


( B - D ) HeLa cells treated with control , PI5P4K2A , 2B , and 2C siRNA were transfected with GFP - WIPI2B ( B ) , GFP - WIPI2D ( C ) , or GFP - DFCP1 ( D ) ; starved in HBSS ( 1 hr ) ; and then incubated with Wm in HBSS . WIPI2 or DFCP1 structures were tracked and quantified as in ( A ) .

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(B-D) HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA were transfected with GFP-WIPI2B (B), GFP-WIPI2D (C), or GFP-DFCP1 (D); starved in HBSS (1 hr); and then incubated with Wm in HBSS. WIPI2 or DFCP1 structures were tracked and quantified as in (A).


( E ) Lysates from HeLa cells stably expressing GFP - WIPI2B were incubated with agarose beads coated with PI , PI ( 5 ) P , and PI ( 3 ) P , eluted with SDS - PAGE sample buffer , and recovered proteins were assessed by western blotting using antibodies against GFP . Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls .

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

(E) Lysates from HeLa cells stably expressing GFP-WIPI2B were incubated with agarose beads coated with PI, PI(5)P, and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls.


( F - H ) Cell extracts from HeLa cells stably expressing GFP - WIPI2B were incubated for 3 hr with PI ( 5 ) P - containing liposomes ( F and H ) or PI ( 3 ) P - containing liposomes ( G and H ) before a pull - down experiment using the indicated beads . PS - containing liposomes were used as internal controls for both competition assays in ( F and G ) .

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(F-H) Cell extracts from HeLa cells stably expressing GFP-WIPI2B were incubated for 3 hr with PI(5)P-containing liposomes (F and H) or PI(3)P-containing liposomes (G and H) before a pull-down experiment using the indicated beads. PS-containing liposomes were used as internal controls for both competition assays in (F and G).


( I ) Lysates from HeLa cells stably expressing GFP - WIPI2B WT and GFP - WIPI2B FTTG mutant were incubated with agarose beads coated with PI ( 5 ) P and PI ( 3 ) P , eluted with SDS - PAGE sample buffer , and recovered proteins were assessed by western blotting using antibodies against GFP . See also Figure S6 .

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

(I) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5)P and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.


( A ) HeLa cells stably expressing GFP - LC3 were shifted to starvation media ( HBSS or glucose - free DMEM ) for 4 hr ( in the presence of 200 nM Wm or 100 nM YM - 201636 ) . ( B ) Quantification of numbers of GFP - LC3 vesicles per cell in HeLa cells stably expressing GFP - LC3 treated as in ( A ) is shown in the graph ( mean ± SEM ) .

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(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM).


( C ) Western blot analysis of LC3 - II and tubulin levels in MEF and SKNSH cells were treated as in ( A ) in complete media or glucose - free media in the presence of 100 nM YM - 201636 , 200 nM Wm , and 400 nM BAF for 4 hr .

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(C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr.


( D ) HeLa cells transfected with GFP - PHD3X and Strawberry - ATG16L1 for 16 hr were left in complete media ( basal ) or glucose - free media ( glucose free ) for 4 hr in the presence or absence of 100 nM YM - 201636 , then fixed and imaged on a confocal microscope .

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(D) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope.


( E and F ) HeLa cells transfected with GFP - PI5P4K2A , 2B , 2C , or PI5P4K2C catalytic dead and RFP - LC3 for 30 hr ( E ) were starved for glucose for 4 hr , then fixed and imaged on a confocal microscope . Bar , 10 μm . ( F ) Quantification of cells ( percentage of total ) showing more than ten autophagic vesicles ( RFP - LC3 vesicles ) in the different conditions from ( F ) is shown in the graph ; n = 200 cells ( mean ± SEM ) .

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(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM).


( G ) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP - PHD3X , Strb - ATG16L1 , and myc - MTMR3 WT . Cells were starved for glucose for 4 hr , then fixed , stained with anti - myc antibodies , and imaged on a confocal microscope . Bar , 10 μm .

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(G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm.


( H ) HeLa cells transfected for 16 hr with GFP - PHD3X and Strb - ATG16L1 were starved for 4 hr in HBSS or glucose - free media , then fixed , stained for PI ( 3 ) P antibodies , and imaged on a confocal microscope . Bar , 10 μm . See also Figure S7 .

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(H) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3)P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.


( A ) Flow cytometry analysis of l ( 2 ) mbn cells starved for 2 h ( 2h S ) or 4 h ( 4h S ) showed an increase in LTG fluorescence levels ( x axis ) compared with control cells in full - nutrient medium ( C ) . The gate shown on the histogram represents the LTGhigh population . ( B ) Representative images of GFP - LC3 puncta in control and 2 - h starved l ( 2 ) mbn cells . An increase in GFP - LC3 puncta was observed in the starved cells ( left ) . Bar , 10 μm . ( C ) Flow cytometry analysis of 4 - h starved cells were incubated with 3MA ( 4h S + 3MA ) and Baf ( 4h S + Baf ) . Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells ( 4h S ) . Control cells in nutrient - full medium ( C ) are represented by the brown line . ( D ) Both autophagy inhibitors , 3MA and Baf , reduced the LTGhigh population significantly . ( 3MA , P = 0 . 00001 ; and Baf , P = 0 . 00006 ) .

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

(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).


( E ) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control . ( Atg1 , P = 0 . 01 ; Atg5 , P = 0 . 01 ; Atg7 , P = 0 . 002 ; Atg8a , P = 0 . 008 ; Atg8b , P = 0 . 006 ; and Atg12 , P = 0 . 02 ) .

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(E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02).


( F ) RNAi of all tested genes in the TOR - PI3K pathways had a statistically significant effect on LTG fluorescence levels . Known negative regulators of autophagy are shown with gray bars ; positive regulators are shown with white bars . ( Pten , P = 0 . 007 ; Tsc1 , P = 0 . 027 ; Tsc2 , P = 0 . 025 ; RheB , P = 0 . 005 ; Tor , P = 0 . 016 ; and S6k , P = 0 . 012 ) . dsRNA corresponding to a human gene ( Hs ) was used as a negative control in E and F . Results represent the mean value ± SD from at least three independent experiments .

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(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.


( A ) The percentage of LTGhigh cells was reduced by hid - RNAi ( P = 0 . 006 ) but not by rpr , grim , and skl - RNAi .

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(A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi.


( B ) Knockdown of Ras , phl , and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells . ( Ras , P = 0 . 003 ; phl , P = 0 . 001 ; and rl , P = 0 . 028 ) .

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(B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028).


( C ) th - RNAi treatment ( 24 h ) had no significant effect on LTG levels ; in contrast , RNAi of Bruce resulted in an increase in LTG fluorescence levels ( P = 0 . 01 ) .

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(C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01).


( D ) Reduction of debcl , Buffy , or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels . ( debcl , P = 0 . 018 ; Buffy , P = 0 . 006 ; and p53 , P = 0 . 004 ) .

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(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).


( E ) RNAi of effector caspase Dcp - 1 resulted in a significant decrease in the LTGhigh population ( P = 0 . 001 ) .

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(E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001).


( F ) Representative images of GFP - LC3 puncta in cells treated with the indicated RNAi after a 2 - h starvation treatment . Bar , 10 μm .

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(F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm.


( G ) Quantification of cells with GFP - LC3 puncta after RNAi treatment . Cells with more than three GFP - LC3 punctate dots were considered to be GFP - LC3 - positive cells . Cells treated with the RNAi indicated here all showed a significant difference ( P < 0 . 05 ) in the percentage of GFP - LC3 - positive cells compared with the human ( Hs ) RNAi control . ( Pten , P = 0 . 006 ; Tor , P = 0 . 034 ; Buffy , P = 0 . 005 ; debcl , P = 0 . 003 ; Bruce , P = 0 . 003 ; Dcp - 1 , P = 0 . 007 ; hid , P = 0 . 002 ; Ras , P = 0 . 006 ; and phl , P = 0 . 050 ) . Results represent the mean value ± SD from three independent experiments .

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(G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.


( A ) GFP - LC3 proteins were expressed in nurse cells ( NC ) but not in follicle cells ( FC ) by using the UASp / nanos Gal4 system . DAPI staining of nuclei is shown in blue .

(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue.


( B ) UASp - GFP - LC3 ; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP - LC3 pattern . Numerous GFP - LC3 puncta ( green ) at region 2 within germarium were observed in nutrient - deprived flies . Ovaries were stained with LTR in w1118 flies . Germarium of nutrient - deprived w1118 flies had an increase in punctate LTR staining ( red ) compared with well - fed germarium .

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(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.


( C ) Degenerating stage 8 egg chambers ( arrows ) had numerous GFP - LC3 puncta ( green ) and an increase in LTR - positive dots ( red ) compared with healthy egg chambers ( arrowheads ) . DAPI ( blue ) staining of nuclei is shown in the two panels on the right .

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(C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right.


( D ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) showed a dramatic decrease in LTR staining . DAPI staining of nuclei is shown in blue .

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(D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.


( A ) Germaria of the nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 B . DAPI staining of nuclei is shown in blue . ( B ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 C .

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(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.


( C ) Lack of Dcp - 1 function ( UASp - GFP - LC3 Dcp - 1Prev / Dcp - 1Prev ; nanos - GAL4 / + ) resulted in uniform diffuse staining of GFP - LC3 rather than the punctate pattern observed in wild - type degenerating stage 8 egg chambers shown in Fig 3 C . ( D ) Dying egg chambers ( arrows ) of NGT / + ; nanos - GAL4 / UASp - fl - Dcp - 1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining ( red ) compared with healthy egg chambers ( arrowheads ) . ( E ) Expression of activated Dcp - 1 ( a truncated form ) and GFP - LC3 in the germ line ( UASp - GFP - LC3 / + ; nanos - GAL4 / nanos - GAL4 UASp - tDcp - 1 ) resulted in abundant degenerating stage 8 egg chambers ( arrows ) with numerous GFP - LC3 puncta ( green ) . DAPI staining of nuclei is shown in blue .

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(C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue.


( A ) The germarium in well - fed BruceE81 flies showed an increase in LTR staining ( red ) compared with wild - type well - fed flies shown in Fig . 3 B ( top right ) . DAPI staining ( white ) of nuclei is shown on the right . ( B ) In well - fed wild - type flies , mid - oogenesis nurse cell death is a rare event . Lack of Bruce function resulted in an increase in dying stage 8 egg chambers ( arrows ) in ovaries under well - fed conditions , and these degenerating stage 8 egg chambers had numerous LTR ( red ) punctate dots . DAPI staining ( white ) is shown on the right . Bars : ( A ) 20 μm ; ( B ) 50 μm .

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(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.


( A ) Ovaries were stained with TUNEL ( green ) to detect DNA fragmentation . Clusters of cysts with TUNEL staining were observed in region 2 in nutrient - deprived w1118 files . In Dcp - 1Prev flies , fewer TUNEL - positive cysts in region 2 were observed . Under well - fed conditions , numerous TUNEL - positive cysts were observed in BruceE81 flies . DAPI staining of nuclei is shown in white . ( B ) Numerous degenerating stage 8 egg chambers ( arrows ) with Bruce - positive staining ( green ) were observed in well - fed BruceE81 flies . DAPI staining of nuclei ( white ) is shown on the right .

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(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.


( A ) TUNEL - positive staining was observed in dying stage 8 egg chambers ( arrows ) of starved control flies ( CG5335d30 / Atg7d14 ) . DAPI staining of nuclei ( white ) is shown on the right . ( B ) In nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation , detected by DAPI , was still observed . ( C ) Dying stage 8 egg chambers ( arrows ) from nutrient - deprived control siblings ( Atg1Δ3D / TM3 ) generated from the same cross in D had abundant TUNEL - positive staining . ( D ) In nutrient - deprived Atg1 GLCs , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation ( DAPI , right ) in degenerating egg chambers appeared to occur as in the controls .

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(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.


B : U2 - OS cells were transfected with the indicated siRNAs . The indicated cells were treated with etoposide ( 50 µM , 90 min . before analysis ) . Whole - cell extracts were immunoblotted with the indicated antibodies . The result is representative of three biological replicates .

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B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.


D : U2 - OS cells were transfected with non - targeting ( mock ) or TSG101 - targeting siRNAs and irradiated ( 20 Gy ) 90 min . before analysis . Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one - way ANOVA ( * p < 0 . 05 ) in the right panel .

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D: U2-OS cells were transfected with non-targeting (mock) or TSG101-targeting siRNAs and irradiated (20 Gy) 90 min. before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one-way ANOVA (*p < 0.05) in the right panel.


A : Nuclear - cytoplasmic fractionation of U2 - OS cells . Indicated samples were transfected with siRNAs against TSG101 . PARylation was inhibited by olaparib ( 10 µM , 24 hours before analysis ) . Cytoplasmic export of p - ATM was induced by etoposide ( 50 µM , 45 minutes before analysis ) . Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies . The fractionation efficiency was controlled by the respective subcellular marker proteins ( PARP1 and LDH - A ) . PARylation was detected using the Pan ADPr reagent . Note that PARylation is activated by shearing forces during extract preparation ( lane 1 )

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A: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1)


B : U2 - OS cells were transfected with non - targeting ( - ) or TSG101 - targeting ( + ) siRNAs and cells were irradiated ( + ) or not ( - ) as indicated ( 20 Gy ) 10 min . before analysis . Whole - cell extracts were immunoblotted with the 10H PAR monoclonal antibody .

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B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.


C : siRNA - transfected U2 - OS cells as in B were etoposide - treated or not , as indicated . Whole - cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly - and mono - ( ADP - ribose ) . Note that PARylation also occurred during cell lysis in untreated cells .

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C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.


D : U2 - OS cells were transfected with non - targeting or VPS28 - targeting siRNAs . Whole - cell extracts were immunoblotted with indicated antibodies .

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D: U2-OS cells were transfected with non-targeting or VPS28-targeting siRNAs. Whole-cell extracts were immunoblotted with indicated antibodies.


F : Cells were irradiated ( 20 Gy ) as indicated 10 min before analysis . Whole - cell extracts were immunoblotted with the indicated antibodies .

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F: Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole-cell extracts were immunoblotted with the indicated antibodies.


A : Proximity ligation assay ( PLA ) assay with unstimulated , etoposide ( Eto ) - treated ( 50 µM , 10 min . before analysis ) , or etoposide plus olaparib - ( Olap ) co - treated ( 50 µM of etoposide , 10 min . before analysis and 10 µM of olaparib 24 hours before analysis ) U2 - OS cells . The red dots represent PARP1 - TSG101 interaction . The nucleus is stained with DAPI ( blue ) . Scale bar : 10 µm . As negative controls , PLA was performed without primary PARP1 or TSG101 antibodies . As an additional control , PLA was performed in PARP1 depleted cells to show specificity B : Quantification of PLA dots per nuclei in A . Blind counting was performed from 30 replicates three biologically independent experiments . The conditions were compared with an ordinary one - way ANOVA ( ns , p > 0 . 05 ; * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001).


C : U2 - OS cells were cotransfected with the indicated deletion constructs of mCherry - tagged TSG101 and EGFP - tagged PARP1 . Immunoprecipitation of GFP was performed using whole - cell extracts .

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C: U2-OS cells were cotransfected with the indicated deletion constructs of mCherry-tagged TSG101 and EGFP-tagged PARP1. Immunoprecipitation of GFP was performed using whole-cell extracts.


F : In - vitro PARylation reactions were carried out with the indicated recombinant and purified proteins , including 8 ng PARP1 in the presence of sssDNA ( sheared salmon sperm DNA ) and MgCl2 . 100 ng of TSG101 was added to the reaction , as indicated . The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls .

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F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.


B : Representative images of PARP1 - GFP association with laser - microirradiation sites in untreated ( mock ) , olaparib - treated ( 10 µM , 24 hours before analysis ) or siTSG101 - transfected U2 - OS cells at indicated times post - irradiation . Scale bar : 7 . 5 µm . The image is representative of nine replicates from three biologically independent experiments . Go to Movies EV1 - 3 for further illustration .

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B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.


C : Kinetics of PARP1 - GFP recruitment and dissociation from DNA lesions were measured at times indicated . Nine nuclei were analyzed for each indicated condition from three biologically independent experiments . The data are shown as mean GFP intensity in the microirradiated area ± SEM , normalized to the mean GFP intensity of corresponding whole nuclei . D : Relative PARP1 - GFP recruitment to DNA lesions for each indicated condition at 12 min . post - irradiation .

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C: Kinetics of PARP1-GFP recruitment and dissociation from DNA lesions were measured at times indicated. Nine nuclei were analyzed for each indicated condition from three biologically independent experiments. The data are shown as mean GFP intensity in the microirradiated area ± SEM, normalized to the mean GFP intensity of corresponding whole nuclei. D: Relative PARP1-GFP recruitment to DNA lesions for each indicated condition at 12 min. post-irradiation.


C : Cells were transfected with non - targeting or TSG101 - targeting siRNAs . Immunofluorescence staining was performed with the indicated antibodies ( red : cleaved caspase 3 ; green : γH2AX ) . Nuclei were stained with DAPI ( blue ) . The image is a representative of 3 biologically independent experiments . Scale bar : 30 µm . See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR - treatment . D : Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments . Conditions were compared with an unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment. D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01).


E : γH2AX staining of TSG101 - targeting or non - targeting siRNA transfected U2 - OS cells reveals puncta - like foci at 2 or 7 days after irradiation .

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E: γH2AX staining of TSG101-targeting or non-targeting siRNA transfected U2-OS cells reveals puncta-like foci at 2 or 7 days after irradiation.


F : The number of puncta - like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments .

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F: The number of puncta-like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments.


( A ) In situ hybridizations for Elp3 transcripts in the developing inner ear . Transcripts are present at E12 . 5 in a ventro - medial region of the developing cochlear duct ( CD ) and in the cochleo - vestibular ganglion ( CVG ) . At E16 . 5 , transcripts are present in the spiral ganglion neurons ( SGNs ) and in the newly formed hair cells ( HCs ) at the base of the cochlea , where they remain strongly expressed at P1 . Scale bar = 50µm ; inset = 25µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti .

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(A) In situ hybridizations for Elp3 transcripts in the developing inner ear. Transcripts are present at E12.5 in a ventro-medial region of the developing cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG). At E16.5, transcripts are present in the spiral ganglion neurons (SGNs) and in the newly formed hair cells (HCs) at the base of the cochlea, where they remain strongly expressed at P1. Scale bar=50µm; inset=25µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.


( B ) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth . Scale bar = 10µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 , OC : Organ of Corti .

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(B) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar=10µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.


Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( C ) At P1 , Elp3 mRNA is absent from Elp3cKO . Scale bars = 50µm . In situ hybridizations were repeated 4 times .

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Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (C) At P1, Elp3 mRNA is absent from Elp3cKO. Scale bars=50µm. In situ hybridizations were repeated 4 times.


Validation of Elp3 depletion from the cochlea in Elp3cKO animals . ( D ) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level ( cochleae from 3 different animals are pooled per sample ) .

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Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (D) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level (cochleae from 3 different animals are pooled per sample).


( E ) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound - evoked potentials in Elp3cKO animals , indicating that they suffer from severe hearing loss . ( F ) Elp3cKO animals present increased ABR thresholds ( WT / Elp3cKO n = 5 / 3 . Unpaired two - tailed t - test , * * p = 0 . 0017 , t = 5 . 369 , DF = 6 , mean ± SD ) .

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(E) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound-evoked potentials in Elp3cKO animals, indicating that they suffer from severe hearing loss. (F) Elp3cKO animals present increased ABR thresholds (WT/Elp3cKO n=5/3. Unpaired two-tailed t-test, **p=0.0017, t=5.369, DF=6, mean±SD).


( A ) Toluidine blue stainings of semi - thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss . Scale bar = 100µm , OC : Organ of Corti .

(A) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti.


( B ) Specific labellings of the pre - synaptic marker Ctbp2 ( in IHCs ) and the post - synaptic marker GluR2 / 3 ( in SGN terminals ) indicate a decrease in the number of double - labelled dots in P15 Elp3cKO whole - mounted cochlea compared to WT . The remaining synapses still show coupling of the pre - and post - synaptic markers , suggesting they may be functional . Scale bar = 20µm . ( C ) The mean number of Ctbp2 - GluR2 / 3 double - positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65 % compared to control ( n = 6 animals per genotype ; Unpaired two - tailed t - test , * * * p < 0 . 001 , t = 13 . 48 , DF = 10 , mean ± SD ) .

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(B) Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar=20µm. (C) The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n=6 animals per genotype; Unpaired two-tailed t-test, ***p<0.001, t=13.48, DF=10, mean±SD).


( D ) NF - 1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae . Scale bar = 100µm .

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(D) NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar=100µm.


( E ) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14 . 5 onwards ( n = 3 - 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * p < 0 . 05 ; F = 20 . 43 ; DF = 7 ; mean ± SD ) .

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(E) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14.5 onwards (n=3-6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; *p<0.05; F=20.43; DF=7; mean±SD).


( F ) Detection of apoptotic SGNs from E13 . 5 cochlear sections with cleaved caspase - 3 and B - tubIII stainings . Scale bar = 100µm .

(F) Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar=100µm.