( G , H ) The replication of EMCV in WT ( G ) and ENT3 - / - BMDMs ( H ) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 9 hours post - infection ( MOI = 10 ) . The expression of EMCV - 2A2B was determined by RT - qPCR . Shown are results from three to four biological replicates ( n = 3 - 4 ) . Data information : In G , H ) the EMCV - 2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1 . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM .

O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) The replication of EMCV in WT (G) and ENT3-/- BMDMs (H) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 9 hours post-infection (MOI=10). The expression of EMCV-2A2B was determined by RT-qPCR. Shown are results from three to four biological replicates (n=3-4). Data information: In G, H) the EMCV-2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1. Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.


( A ) Illustration of strategy to track the viral uncoating upon infection . ( B - D ) ( B ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and subjected to live imagining 30 , 60 and 90 minutes post - infection . The arrowheads indicated the Syto82 + virus puncta in the cell . Scale bars represent 10 µm . ( C ) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82 + cells ( % ) and ( D ) the mean fluorescent intensity ( MFI ) per Syto82 + cell ( D ) . Results were generated from images of three biological replicates ( n = 3 ) , each with 4 fields analyzed . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Illustration of strategy to track the viral uncoating upon infection. (B-D) (B) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and subjected to live imagining 30, 60 and 90 minutes post-infection. The arrowheads indicated the Syto82+ virus puncta in the cell. Scale bars represent 10 µm. (C) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82+ cells (%) and (D) the mean fluorescent intensity (MFI) per Syto82+ cell (D). Results were generated from images of three biological replicates (n=3), each with 4 fields analyzed. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( E ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and co - stained with 100 nM Lysotracker Green , subjected to live imagining 60 minutes post - infection . Data were the representative images of two biological replicates ( n = 2 ) . Scale bars represent 5 µm . ( F ) WT and ENT3 - / - BMDMs were infected with EMCV ( MOI = 10 ) for 2 hours and then enriched the endosomes and lysosomes . EMCV particles in endosome - or lysosome - enriched fractions were quantified by plaque assay . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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(E) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and co-stained with 100 nM Lysotracker Green, subjected to live imagining 60 minutes post-infection. Data were the representative images of two biological replicates (n=2). Scale bars represent 5 µm. (F) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10) for 2 hours and then enriched the endosomes and lysosomes. EMCV particles in endosome- or lysosome-enriched fractions were quantified by plaque assay. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( G - K ) ( G ) Calu - 3 cells were infected with SARS - CoV - 2 original strain or delta variant at MOI = 0 . 1 for 48 hours , harvested , and examined for SLC29A3 induction . ( H ) ENT3 knockdown in Calu - 3 cells was performed by lentiviral shRNA system . The knockdown efficiency was confirmed by RT - qPCR . ENT3 knockdown Calu - 3 cells were infected with SARS - CoV - 2 delta variant and subjected for the viral replication analysis 24 hours post - infection . The virus released out of cells was measured by plaque assay ( I ) , and the viral titer in the cells was measured by RT - qPCR ( J ) . ( K ) The replication of SARS - CoV - 2 delta variant in scramble and ENT3 knockdown Calu - 3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 24 hours post - infection ( MOI = 0 . 1 ) . The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1 . The SARS - CoV - 2 E gene expression level was calculated relative to RPLP0 to normalize the cell number . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-K) (G) Calu-3 cells were infected with SARS-CoV-2 original strain or delta variant at MOI=0.1 for 48 hours, harvested, and examined for SLC29A3 induction. (H) ENT3 knockdown in Calu-3 cells was performed by lentiviral shRNA system. The knockdown efficiency was confirmed by RT-qPCR. ENT3 knockdown Calu-3 cells were infected with SARS-CoV-2 delta variant and subjected for the viral replication analysis 24 hours post-infection. The virus released out of cells was measured by plaque assay (I), and the viral titer in the cells was measured by RT-qPCR (J). (K) The replication of SARS-CoV-2 delta variant in scramble and ENT3 knockdown Calu-3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 24 hours post-infection (MOI=0.1). The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1. The SARS-CoV-2 E gene expression level was calculated relative to RPLP0 to normalize the cell number. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Western blotting showing the expression of RAP1 and TRF2 in MRC - 5 cells of different population doublings ( PD ) . Senescent cells ( PD 72 ) were left in culture for at least 4 weeks before harvesting for analysis .

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Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis.


ChIP analysis of young ( PD 30 ) and senescent ( PD 72 ) MRC - 5 cells performed with either anti - RAP1 , anti - TRF2 or an IgG antibody . The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe , stripped and hybridized to an Alu probe in order to determine unspecific binding . Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input . Data represent mean + / - SD of three biological replicates .

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ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2 or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean +/- SD of three biological replicates.


Immunofluorescence detection of 53BP1 ( red ) and a FISH probe staining telomeres ( green ) in young ( PD 26 ) , pre - senescent ( PD 66 ) and senescent ( PD 72 + 4 weeks ) MRC - 5 fibroblasts . The upper graph shows the percentage of telomere co - localizing with 53BP1 ( TIFs ) . Total DNA damage was measured by immunofluourescence detection of 53BP1 . The lower graph shows the total number of 53BP1 foci per nucleus . Approximately 40 - 50 cells were analyzed per replicate and per condition . Bars represent SEM of 2 biological replicates .

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Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66) and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluourescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40-50 cells were analyzed per replicate and per condition. Bars represent SEM of 2 biological replicates.


Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction ( 16p1 , 21q1 and XpYpM ) . The primer 16p1 is able to bind the subtelomeric region of 1p 9q , 12p , 15q , 16p , XqYq and the interstitial 2q14 region ; the 21q1 primer binds the subtelomeric region of 1q , 2q , 5q , 6q , 6p , 8p , 10q , 13q , 19p , 19q , 21q , 22q and the interstitial 2q13 site . The PCR - fusion assay was performed in MRC - 5 primary fibroblasts of different population doublings ( PD ) with or without depletion of RAP1 ( shRAP1 or shControl respectively ) . Lentiviral infection was performed for 10 days . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * * p < 0 . 001 ; * * * p < 0 . 0001 ) . Southern blotting showing the hybridization of the 16p probe for the conditions described in A . Number of fusions per 1 µg of DNA is shown at the bottom of the gels .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O

Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction (16p1, 21q1 and XpYpM). The primer 16p1 is able to bind the subtelomeric region of 1p 9q, 12p, 15q, 16p, XqYq and the interstitial 2q14 region; the 21q1 primer binds the subtelomeric region of 1q, 2q, 5q, 6q, 6p, 8p, 10q, 13q, 19p, 19q, 21q, 22q and the interstitial 2q13 site. The PCR-fusion assay was performed in MRC-5 primary fibroblasts of different population doublings (PD) with or without depletion of RAP1 (shRAP1 or shControl respectively). Lentiviral infection was performed for 10 days. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (**p < 0.001; ***p < 0.0001). Southern blotting showing the hybridization of the 16p probe for the conditions described in A. Number of fusions per 1 µg of DNA is shown at the bottom of the gels.


Number of fusions per 1 µg of DNA in MRC - 5 senescent cells . Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days ( two subsequent transfections of 3 days each ) . Representative southern blot membranes hybridized with the 16p probe are shown . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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Number of fusions per 1 µg of DNA in MRC-5 senescent cells. Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days (two subsequent transfections of 3 days each). Representative southern blot membranes hybridized with the 16p probe are shown. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).


Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1 . Variant telomere repeats were included in the estimation of the telomere array length . Data represent median + / - interquartile range ( green lines ) of 3 biological replicates . Statistical analysis was performed using Mann - Whitney U test * * * p < 0 . 0001 ) .

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Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1. Variant telomere repeats were included in the estimation of the telomere array length. Data represent median +/- interquartile range (green lines) of 3 biological replicates. Statistical analysis was performed using Mann-Whitney U test ***p < 0.0001).


RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days ( 20 µM final concentration ) . Fifteen days before cell harvesting , doxycycline ( DOX ; 1 µg / µl final concentration ) was added to deplete the expression of RAP1 .

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RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1.


Telomere length analysis by southern blotting of the samples described in A . The size of the main intensity peak is indicated at the bottom of the gel .

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Telomere length analysis by southern blotting of the samples described in A. The size of the main intensity peak is indicated at the bottom of the gel.


Number of fusions in HeLa cells after 25 days in culture . Cells were maintained with BIBR1532 during the whole period of the experiment , while doxycycline ( 1 µg / µl final concentration ) and the infection with shControl , shLIG3 or shLIG4 was carried out for the last 15 days of the experiment . Data represent mean + / - SD of three biological replicates are shown . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).


Examples of metaphase spreads hybridized with a telomeric PNA probe ( green ) when RAP1 was depleted ( + DOX ) . Telomerase was inhibited with BIBR1532 for 25 days . Scale bar = 10 µm . Quantification of chromosome aberrations observed in RAP1 depleted cells ( + DOX ) or control ( - DOX ) . Data represent mean + / - SD of three biological replicates . Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition . * p < 0 . 05 ; two - tailed Student ' s t test .

O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm. Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.


Growth curve of MRC - 5 fibroblasts . Cells were grown until senescence and after three weeks of no growth detection , lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed . Cells were harvested 15 days post infection ( dpi ) . Data represent mean + / - SD of three biological replicates measured after 8 and 15 dpi .

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Growth curve of MRC-5 fibroblasts. Cells were grown until senescence and after three weeks of no growth detection, lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed. Cells were harvested 15 days post infection (dpi). Data represent mean +/- SD of three biological replicates measured after 8 and 15 dpi.


Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl , shp21CIP1 or shp21CIP1 + shRAP1 .

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Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1.


Dividing cells ( magenta ) were identified by EdU staining ( 1 µM EdU for 24 h ) . The percentage of dividing cells after 15 dpi is shown ( n = 3 ) . Scale bar = 10 µm .

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Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm.


Senescence - associated β - galactosidase ( SA - β - gal ) staining in senescent and post - senescent fibroblasts ( 1 and 15 dpi ) is shown . Percentage of SA - β - gal positive cells is indicated for each condition . Three biological replicates were performed , with approximately 300 cells analyzed per condition . Scale bar = 50 µm .

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Senescence-associated β-galactosidase (SA-β-gal) staining in senescent and post-senescent fibroblasts (1 and 15 dpi) is shown. Percentage of SA-β-gal positive cells is indicated for each condition. Three biological replicates were performed, with approximately 300 cells analyzed per condition. Scale bar = 50 µm.


Senescent cells , transduced with shp21CIP1 or shp21CIP1 + shRAP1 for 15 days , were stained with CytoCalcein violet ( live cells ) , Apopxin Green ( apoptotic cells ) and 7 - aminoactinomycin D ( 7 - AAD ) ( late apoptostic / necrotic cells ) and visualized by flow cytometry . Quantification of apoptotic cells from the conditions described in E . Data represent mean + / - SD of three biological replicates ( * * P < 0 . 001 ; two - tailed Student ' s t test ) .

B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry. Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).


ATAC - seq peaks more accessible in GPR56low ( violet ) or GPR56high ( turquoise ) against the background ( grey ) are enriched for enhancers associated with specific hematopoietic cell types . P - values and odds ratios are given for a pairwise , two - sided Fisher ' s Exact test comparing each category ( GPR56low / high ) against the background . Enhancer annotations are taken from EnhancerAtlas 2 . 0 . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ATAC-seq peaks more accessible in GPR56low (violet) or GPR56high (turquoise) against the background (grey) are enriched for enhancers associated with specific hematopoietic cell types. P-values and odds ratios are given for a pairwise, two-sided Fisher's Exact test comparing each category (GPR56low / high) against the background. Enhancer annotations are taken from EnhancerAtlas 2.0. *** p<0.0005, ** p<0.005, * p<0.05.


Knockdown efficiency of two shRNAs against GPR56 ( shGPR56weak and shGPR56strong ) versus shLuc as negative control measured on protein level by flow cytometry in CD34 + CB cells . Shown are representative FACS plots ( left ) and the percentage of GPR56 + cells of transduced GFP + cells on day 5 ( right panel ) . Biological N = 4 , unpaired t - test , bars and error bars represent mean and standard deviation . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE B-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Knockdown efficiency of two shRNAs against GPR56 (shGPR56weak and shGPR56strong) versus shLuc as negative control measured on protein level by flow cytometry in CD34+ CB cells. Shown are representative FACS plots (left) and the percentage of GPR56+ cells of transduced GFP+ cells on day 5 (right panel). Biological N=4, unpaired t-test, bars and error bars represent mean and standard deviation. *** p<0.0005, ** p<0.005, * p<0.05.


Integrative Genome Viewer ( IGV ) snapshot showing ATAC - seq peaks along and upstream of the VWF gene in GPR56high vs . low samples ( top , average peak size of 10 GPR56high ( turquoise ) and 15 GPR56low samples ( violet ) ) , RNA - seq reads of the same location in AML samples with high ( n = 9 ) versus low ( n = 11 ) GPR56 expression ( two middle tracks ) , and RNA - seq reads of shLuc versus GPR56 knockdown CD34 + cells ( 3 bottom tracks , one of two replicates shown for each condition ) . Track height was group - scaled . Dashed vertical lines indicate binding sites for the annotated TFs . TFBS : transcription factor binding site derived from the HOCOMOCO v10 database ; TSS : transcription start site . TFs in blue bind to differentially accessible chromatin regions .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-DISEASE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O 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-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O

Integrative Genome Viewer (IGV) snapshot showing ATAC-seq peaks along and upstream of the VWF gene in GPR56high vs. low samples (top, average peak size of 10 GPR56high (turquoise) and 15 GPR56low samples (violet)), RNA-seq reads of the same location in AML samples with high (n=9) versus low (n=11) GPR56 expression (two middle tracks), and RNA-seq reads of shLuc versus GPR56 knockdown CD34+ cells (3 bottom tracks, one of two replicates shown for each condition). Track height was group-scaled. Dashed vertical lines indicate binding sites for the annotated TFs. TFBS: transcription factor binding site derived from the HOCOMOCO v10 database; TSS: transcription start site. TFs in blue bind to differentially accessible chromatin regions.


Volcano plot of differential transcription factor ( TF ) motif accessibility ( activity ) in GPR56high ( turquoise ) vs GPR56low ( violet ) samples and their corresponding adjusted p - values determined with diffTF . Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA - seq dataset .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O

Volcano plot of differential transcription factor (TF) motif accessibility (activity) in GPR56high (turquoise) vs GPR56low (violet) samples and their corresponding adjusted p-values determined with diffTF. Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA-seq dataset.


Violin plots showing geometric mean ( horizontal line ) and individual values ( circles ) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34 + cells following overnight - infection with shGPR56strong ( turqouise ) and shGPR56weak ( gray ) versus shLuc control ( violet ) . Shown are percentages of indicated populations , which are also co - positive for human CD45 and GFP , relative to all harvested bone marrow cells . HSPC : CD34 + SSClowCD33 - CD19 - , B precursor : CD34 + CD19 + CD33 - , myeloid progenitor ( myelo . prog . ) : CD34 + CD33 + CD19 - , mature B cells : CD19 + CD34 - , mature myeloid cells : CD33 + CD19 - CD34 - . Multiple unpaired t - tests of log - transformed values , p - values were Benjamini and Hochberg corrected ( padj ) , * padj < 0 . 05 , * * padj < 0 . 005 , * * * padj < 0 . 0005 .

O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O 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-ORGANISM B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots showing geometric mean (horizontal line) and individual values (circles) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34+ cells following overnight-infection with shGPR56strong (turqouise) and shGPR56weak (gray) versus shLuc control (violet). Shown are percentages of indicated populations, which are also co-positive for human CD45 and GFP, relative to all harvested bone marrow cells. HSPC: CD34+SSClowCD33-CD19-, B precursor: CD34+CD19+CD33-, myeloid progenitor (myelo. prog.): CD34+CD33+CD19-, mature B cells: CD19+CD34-, mature myeloid cells: CD33+CD19-CD34-. Multiple unpaired t-tests of log-transformed values, p-values were Benjamini and Hochberg corrected (padj), * padj<0.05, ** padj<0.005, *** padj<0.0005.


Adhesion of control and GPR56 KD K562 cells on fibronectin - functionalized substrates . Left : Overlay of phase contrast and microinterferometry images of control ( above ) and GPR56 KD cells ( below ) . The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet , respectively . Right : Comparison of adhesion areas extracted by microinterferometry , reduction of adhesion area by factor 1 . 9 in GPR56 KD cells ( p = 1 . 0 x 10 - 10 , two - sided Mann - Whitney test , box plots showing medians , quartiles , and outliers according to the Tukey method ) . Technical replicates Ncontrol = 71 , Technical replicates NKD = 104 , scale bar 10 µm .

O O O O B-GENEPROD O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Adhesion of control and GPR56 KD K562 cells on fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm.


β - catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a ( representative Western Blot ( above ) and quantification after normalization to GAPDH ( below ) . Shown is fold - change compared to shLuc in control media , bars and error bars represent mean and standard deviation of three biological replicates , unpaired t - test ) .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

β-catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a (representative Western Blot (above) and quantification after normalization to GAPDH (below). Shown is fold-change compared to shLuc in control media, bars and error bars represent mean and standard deviation of three biological replicates, unpaired t-test).


Left : Representative immunofluorescence ( IF ) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc ( above ) or shGPR56strong ( below ) . The non - motile primary cilium is visualized using antibodies against γ - tubulin , which stains the basal body , and the ciliary membrane marker ARL13B . DAPI indicates the nucleus . Images on the right show selected cells at 3 x magnification . Brightness was increased in all images to enhance visibility of cilia . Right : Percentage of RPE - 1 cells with primary cilium in shLuc vs . shGPR56strong RPE - 1 cells at different time points after the start of serum starvation . Unpaired t - test , bars and error bars represent mean and stdev of three biological replicate wells . * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The non-motile primary cilium is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1 cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.


( A ) RT - qPCR analysis of NimB1 , NimB2 , NimB3 , NimB4 and NimB5 transcripts , normalized to RpL32 , from carcass , macrophages , fat body and gut of wild type ( w1118 ) L3 wandering larvae . Data are represented as mean ± SD from three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-TISSUE O B-CELL_TYPE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.


( B ) Representative confocal imaging of NimB4 - sGFP macrophages of embryo at stage 16 ; lateral ( left ) and ventral ( right ) views ; Tissues were stained with anti - GFP ( green , corresponding to NimB4 - sGFP ) and anti - SIMU antibodies ( red , corresponding to macrophages ) . The arrows show the presence of NimB4 - sGFP inside the embryonic macrophages . Scale bars = 20

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O

(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm


( A - C ) Representative confocal imaging of CFSE - stained apoptotic bodies ( green ) incubated with secreted NimB4 - RFP ( B , C ) or SPvkg - RFP ( A ) . Apoptotic bodies were pre - incubated in absence ( A , B ) or presence ( C ) of annexin V ( 25 μg / mL ) . Scale bar = 10 μm . ( D ) Quantification of the colocalization of NimB4 - RFP or SPvkg - RFP with the apoptotic bodies in presence or absence of Annexin V , as measured by Pearson ' s correlation coefficient . Values from at least four independent experiments are represented as mean ± SD ( * * P < 0 . 01 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and apoptosis - null NimB4 sk2 ; Df ( 3L ) H99 embryonic macrophages at stage 16 of development ; ventral and lateral view ; Tissues were co - stained with Dcp - 1 ( red , corresponding to apoptotic corpses ) and anti - SIMU ( green , corresponding to macrophages ) antibodies . The arrows show the presence of apoptotic cells inside embryonic macrophages . Scale bars = 20

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O

(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm


( C ) Representative projections from confocal stacks of entire third instar wild type ( w1118 ) and NimB4 sk2 larval brains stained with anti - Draper ( green ) and anti - Dcp1 ( red ) . The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain . Scale bars = 100

O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O

(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and draper macrophages incubated with fluorescently - labeled apoptotic cells ( red , CellTraceTM Red ) on ice for 1h and stained with AlexaFluorTM 488 phalloidin ( green ) . Scale bar : 10 μm . ( B ) Quantification of the number of apoptotic cells ( red ) binding to the macrophages ( green ) . Values from three independent experiments are represented as mean ± SD ( * * * P < 0 . 001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O 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 confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm. (B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( D ) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies . Wild type , NimB4sk2 , NimB4 genomic rescue ( NimB4sk2 , [ NimB4 ] ) , draper and NimC11 , eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ns : not significant ) . ( E ) Ex vivo phagocytosis assay using AlexFluor488 S . aureus Bioparticles . Wild type , NimB4sk2 and NimC11 , eater1 macrophages from L3 wandering were incubated with bioparticles for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative fluorescence microscopy images of wild type ( w1118 ) , NimB4sk2 , draper , eater1 and croquemort △ third instar larvae macrophages stained with LysoTracker Red ( live imaging ) . Overlay of fluorescence and differential interference contrast microscopy ( DIC ) . Scale bar = 10 μm . ( B ) Mean fluorescence intensity after staining with LysoTracker Red ( live confocal imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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 fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Representative transmission electron micrographs of macrophages from wild type ( top , w1118 ) and NimB4sk2 ( bottom ) L3 wandering larvae . Scale bar : 2 μm ( D ) Quantification of the number of vesicles per macrophages in the electron micrographs . Values from at least three independent experiments are represented as mean ± SD ( * * * * P < 0 . 0001 by Mann - Whitney test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).


( E ) Representative confocal imaging of Rab7EYFP immunostaining in wild type ( w1118 ) and NimB4 sk2 macrophages . Tissues were stained with anti - GFP ( red , Rab7 ) , counterstained with phalloidin ( gray ) and DAPI ( blue ) . The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages . Scale bar = 10 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O O O

(E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.


( A ) Representative confocal imaging of localization of Lamp1 - mcherry and LysoTracker Green in wild type ( w1118 ) , NimB4 sk2 and draper △ 5 macrophages ( live imaging ) . The arrows indicate the colocalization ( w1118 ) or the clustering ( NimB4sk2 and draper △ 5 ) of Lamp1 - mcherry and LysoTracker Green signals . Scale bar : 10 μm . ( B ) Quantification of the colocalization of Lamp1 - mcherry with LysoTracker Green , as measured by Pearson ' s correlation coefficient between the two signals . Values from at least five independent experiments are represented as mean ± SD ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo ™ Red . Wild type ( w1118 ) , NimB4sk2 and draper △ 5 macrophages from L3 wandering larvae were incubated with pHrodo ™ Red apoptotic cells for 60 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using pHrodoTM Red S . aureus BioparticlesTM conjugates . Wild type , NimB4sk2 and NimC11 , eater1 Hml ∆ - Gal4 > UAS - GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S . aureus BioparticlesTM for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A - B ) Representative fluorescence microscopy images of wild type ( A ) or NimB4sk2 ( B ) macrophages expressing Rab5 ( middle panel ) or Rab7 ( right panel ) driven by Hml ∆ - Gal4 and stained with the LysoTracker Red ( live imaging ) . Overlay of fluorescence and DIC . Scale bar = 10 μm . ( C ) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae ( confocal live imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Same as ( E ) with amiRhen1 - 14 scions and non - transgenic WT or hen1 - 14 rootstocks . Average relative U6 - normalized band - intensity quantifications from two biological replicates are indicated .

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Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.


Phenotype of pSUC2 : : amiRSUL plants in hst - 1 / - 25 / - 26 backgrounds .

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Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.


RT - qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes . Error - bars : SD . Welch ' s t - test p - values are indicated . n = 5 .

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RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.


amiRSUL northern analysis in input and AGO1 - immunoprecipitated ( AGO1 - IP ) fractions isolated from leaves of pSUC2 : : amiRSUL plants in the specified genotypes . No Ab : No antibody added to the HST input extract ( IP negative control ) . miR165 / 166 and U6 were probed as endogenous controls . AGO1 western analysis and Coomassie blue ( Coom . ) staining of the western blot membrane are provided as controls . amiRSUL band - intensity quantifications , U6 - normalized for input samples , are indicated .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.


amiRSUL northern analysis , in biological duplicates , in scions ( S ) and rootstocks ( R ) in the specified genotypes ' grafting combinations ( amiR : pSUC2 : : amiRSUL ) ( see also Appendix Fig . S12B ) . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated for each tissue .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O B-TISSUE O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.


amiRSUL northern analysis , in biological duplicates , from total RNA extracted from aphids fed on WT or amiR plants , in the specified genetic backgrounds . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O

amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.


Localization of HST : GFP and , as a reference , of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves ( C ) or roots ( D ) . CW : calcofluor white staining . Scale bars : 20 µm .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-TISSUE O O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O

Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.


Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2 : : HST : GFP , pSUC2 : : GFP or pSUC2 : : tmGFP9 . Scale bars : 100 µm . Confocal fluorescence images of the differentiation and division zones of pSUC2 : : GFPhst - 1 or pSUC2 : : tmGFP9hst - 1 root tips . Scale bars : 100 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE I-TISSUE O B-ORGANISM B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE I-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD B-TISSUE I-TISSUE O O O O O O O

Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.


Subcellular localizations of HST : GFP expressed from the UBQ10 promoter ( pUBQ ) in Arabidopsis roots ( i ) . Distinct planes of the same root cells imaged by confocal microscopy are shown ( ii - iii ) . PI : propidium iodide staining . Scale bars : 50µm ( i ) ; 10µm ( ii - iii ) .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).


Subcellular localizations of hst - 3 : GFP expressed from pUBQ in root cells . Distinct planes of the same root cells imaged by confocal microscopy are shown ( i - ii ) . PI : as in ( A ) . Scale bars : 10µm .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.


miR395 northern analysis in scions ( S ) and hyl1 - 2 rootstocks ( R ) , in the indicated genotypes ' combinations , under SO4 - sufficient ( + SO4 ) or SO4 - starved ( - SO4 ) conditions , in two biological replicates ( rep . ) . U6 was probed as an endogenous control . Relative U6 - normalized band - intensity quantifications are indicated for each tissue in - SO4 conditions .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O

miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.


miR160 northern analysis in Meselect - separated leaf vasculature and epidermis in WT versus hst - 1 plants , in three biological replicates ( rep . ) . U6 : as in ( A ) . Relative U6 - normalized band - intensity quantifications are indicated .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O

miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.


Basic fuchsin staining of protoxylem ( unfilled arrowheads ) and metaxylem ( filled arrowheads ) in WT , shr - 2 or hst - 1 roots . * : protoxylem gap . Scale bars : 10 µm .

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Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.


B . Survival frequencies observed after DSB induction at TEL6R in WT , dnl4Δ , sir4Δ cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . C . Survival frequencies observed after DSB induction at LYS2 in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments. C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


F . Representative images of Mre11 - YFP foci in response to an I - SceI - induced DSB at LYS2 in WT cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Scale bars are 2 μm . G . Quantification of cells with DSB induced Mre11 - YFP foci after DSB induction at LYS2 I - SceI cleavage site in WT , sae2Δ and Sir3 overexpressing ( oeSir3 ) strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm. G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


H . Survival frequencies after DSB induction at LYS2 locus , in strains where SIR3 is expressed from its native , pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid . Fold increase in Sir3 protein by pAHD1 or pGPD is indicated . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Survival frequencies after DSB induction at LYS2 locus, in strains where SIR3 is expressed from its native, pADH1 or pGPD promoters respectively and in which SAE2 is expressed or not from a high copy number 2µ plasmid. Fold increase in Sir3 protein by pAHD1 or pGPD is indicated. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


A . Representative images of Sir3 - mCherry and Sae2 - GFP signal in WT and SIR3 overexpressing cells . Scale bars are 2 μm .

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A. Representative images of Sir3-mCherry and Sae2-GFP signal in WT and SIR3 overexpressing cells. Scale bars are 2 μm.


B . Sir3 - binding at TEL6R in untagged , WT , sir3Δ cells or in cells overexpressing Sir3 ( oeSir3 ) . Binding is probed by ChIP - qPCR 0 . 2 ( red arrows ) and 1kb ( blue arrows ) from telomeres and at the OGG1 control locus using antibodies against Sae2 - GFP . The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates ( SEM ) .

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B. Sir3-binding at TEL6R in untagged, WT, sir3Δ cells or in cells overexpressing Sir3 (oeSir3). Binding is probed by ChIP-qPCR 0.2 (red arrows) and 1kb (blue arrows) from telomeres and at the OGG1 control locus using antibodies against Sae2-GFP. The mean of three independent biological replicates is shown and error bars correspond to the variation between replicates (SEM).


C . Co - immunoprecipitation between Sir3 and Sae2 - GFP from cells overexpressing Sir3 , analysed by Western blot with anti - GFP and anti - Sir3 antibodies . D . Co - immunoprecipitation between Sir3 and Sae2 - GFP from WT cells using antibodies against Sae2 - GFP , analysed by Western blot .

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C. Co-immunoprecipitation between Sir3 and Sae2-GFP from cells overexpressing Sir3, analysed by Western blot with anti-GFP and anti-Sir3 antibodies. D. Co-immunoprecipitation between Sir3 and Sae2-GFP from WT cells using antibodies against Sae2-GFP, analysed by Western blot.


D . Yeast two - hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells . Growth on - His + 3AT and blue coloration on X - gal indicate an interaction .

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D. Yeast two-hybrid interaction analysis between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT and blue coloration on X-gal indicate an interaction.


B . Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C . C . Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C .

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B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.


A . Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival frequencies after DSB induction at LYS2 locus in WT or sae2Δ strains where the Sir3SaID or Sir3SaIDT557I domains are overexpressed from a GPD promoter at the SIR3 locus. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


B . Representative images of Sae2 - GFP in WT cells and in cells overexpressing either full - length Sir3 or the sir3SaID domain . Scale bars are 2 μm .

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B. Representative images of Sae2-GFP in WT cells and in cells overexpressing either full-length Sir3 or the sir3SaID domain. Scale bars are 2 μm.


F . Representative silver - stained gels of in vitro GST - pulldown of GST or GST - Sir3SaID , GST - sir3 - T557ISaID and Sae2C purified peptides . Control : Sae2C ( 300 ng , lane 6 ) .

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F. Representative silver-stained gels of in vitro GST-pulldown of GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).


G . Co - immunoprecipitation between Sae2 - GFP and Sir3 from untagged , Sae2 - GFP WT cells , and Sae2 - GFP cells overexpressing WT Sir3 ( oeSir3 , WT ) , Sae2 - GFP sir3Δ or Sae2 - GFP overexpressing the sir3 - T557I mutant ( oeSir3 , T557I ) using antibodies against Sae2 - GFP , analysed by Western blot with anti - GFP and anti - Sir3 antibodies .

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G. Co-immunoprecipitation between Sae2-GFP and Sir3 from untagged, Sae2-GFP WT cells, and Sae2-GFP cells overexpressing WT Sir3 (oeSir3, WT), Sae2-GFP sir3Δ or Sae2-GFP overexpressing the sir3-T557I mutant (oeSir3, T557I) using antibodies against Sae2-GFP, analysed by Western blot with anti-GFP and anti-Sir3 antibodies.


A . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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A. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


C . Survival frequencies after DSB induction at LYS2 locus in the indicated strains . Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression . Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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C. Survival frequencies after DSB induction at LYS2 locus in the indicated strains. Insertion of the strong TEF1p promoter upstream of the SIR4 ORF leads to Sir4 overexpression. Insertion of the ADH1p promoter upstream of SIR3 leads to mild Sir3 overexpression. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


E . Representative images of two hybrid assays testing the interaction between the full - length Sir3 and full - length Sae2 proteins in WT cells expressing or not high levels of Sir4 ( oeSir4 and WT respectively ) .

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E. Representative images of two hybrid assays testing the interaction between the full-length Sir3 and full-length Sae2 proteins in WT cells expressing or not high levels of Sir4 (oeSir4 and WT respectively).


A . Relative fold enrichment of Sae2 at 0 . 2 kb from I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . B . Relative fold enrichment of Sae2 at 0 . 2 kb from TEL6R after DSB induction at the LYS2 I - SceI site was evaluated by qPCR after ChIP with anti - GFP antibodies . The error bars indicate the variation between at least three biological replicas ( SEM ) . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD 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-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative fold enrichment of Sae2 at 0.2 kb from I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). B. Relative fold enrichment of Sae2 at 0.2 kb from TEL6R after DSB induction at the LYS2 I-SceI site was evaluated by qPCR after ChIP with anti-GFP antibodies. The error bars indicate the variation between at least three biological replicas (SEM). Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


D . Survival frequencies after DSB induction at LYS2 locus in sae2 - E267E mutant cells overexpressing or not full - length Sir3 ( oeSir3 ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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D. Survival frequencies after DSB induction at LYS2 locus in sae2-E267E mutant cells overexpressing or not full-length Sir3 (oeSir3). Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


Schematic , western blot , and immunofluorescence analysis of the 3 × FLAG - UBXD8 knockin U2OS cell line . Scale bar , 5 μm . Data information : In C , mitochondria were visualized by anti - Tom20 immunostaining ; ER was labeled by anti - PDI immunostaining .

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Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm. Data information: In C, mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by anti-PDI immunostaining.


Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆ UBXD8 HeLa cells .

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Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.


Immunoprecipitation analysis of UBXD8 interaction with mitochondrial ( MARCH5 and MUL1 ) and ER ( RNF185 ) ubiquitin E3 ligases . HeLa cells were infected with lentivirus to stably express HA - tagged ubiquitin E3 ligases . Whole cell lysates were prepared for anti - HA immunoprecipitation .

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Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.


Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185 . Whole cell lysates from 3 × FLAG - UBXD8 knockin HEK293T cells were prepared for anti - FLAG immunoprecipitation . MARCH5 and RNF185 : short exposure for whole cell lysate blots , long exposure for FLAG - IP blots .

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Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.


Fractionation analysis of UBXD8 ' s effect on VCP recruitment to mitochondria and the ER . Whole cell lysate , mitochondria , and ER fractions were purified from WT , ∆ UBXD8 , and ∆ UBXD8 HeLa cells rescued with UBXD8 or the UBX * mutant . UBX * - UBXD8 carries four point mutations ( K367A , F407A , P408G , R409A ) in the UBX domain to disrupt the UBXD8 - VCP interaction . For each sample , 5 μg proteins were loaded for western blot analysis .

O O O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.


Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells . Data are shown as mean ± SE from three biological repeats . Statistics : one - way ANOVA ; * * P < 0 . 01 , * * * P < 0 . 001 .

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Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.


A , B . Western blot ( A ) and quantitative ( B ) analyses of MiD49 and Mcl1 degradation in WT and ∆ MARCH5 HeLa cells . Cycloheximide ( CHX : 200 μg / ml ; protein synthesis inhibitor ) ; MG132 : 20 μM , proteasome inhibitor . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


C , D . Western blot ( C ) and quantitative ( D ) analyses of Mcl1 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


E , F . Western blot ( E ) and quantitative ( F ) analyses of MiD49 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats F ) . Statistics : two - tailed unpaired Student ' s t - test F ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats F). Statistics: two-tailed unpaired Student's t-test F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


Fractionation analysis of the subcellular localization of mito - UBXD8 and ER - UBXD8 . Whole cell lysate , mitochondria , and ER fractions were purified from the indicated HeLa cells . Exogenously - expressed mito - UBXD8 has similar expression level with endogenous UBXD8 in WT cells ; exogenously - expressed WT UBXD8 and ER - UBXD8 have similar expression levels .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O O

Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.


D , E . Western blot ( D ) and quantitative ( E ) analyses of Mcl1 degradation in the indicated HeLa cells . ∆ UBXD8 HeLa cells were rescued with vector , WT - , mito - , and ER - UBXD8 . Data are shown as mean ± SE from three biological repeats ( E ) . Statistics : two - tailed unpaired Student ' s t - test ( E ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.


Cell survival analysis of the indicated HeLa cells treated with Actinomycin D ( ActD , 1 μM ) or Doxorubicin ( Doxo , 10 μM ) . Representative images are shown . Cell viability was measured by Cell Titer - Glo . Scale bar , 50 μm . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of apoptosis activation ( caspase cleavage and PARP cleavage ) in the indicated HeLa cells treated similarly as in ( D ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O

Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).