BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml LPS ( A for indicated times . ( A Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml Pam2CSK4 ( B ) B ) Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( C ) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT , MAPK ( ERK , p38 , JNK ) , IκBα , total forms of IκBα . αActin was used as a loading control ( left ) . The densitometric values were calculated from the ratios of protein levels relative to actin ( right ) .

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(C) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT, MAPK (ERK, p38, JNK), IκBα, total forms of IκBα. αActin was used as a loading control (left). The densitometric values were calculated from the ratios of protein levels relative to actin (right).


( A ) Identification of TLR2 , IRAK1 , RIP1 , TRAF6 , MAPK11 and TOLLIP as endogenous binding partners of rRv2626c . THP - 1 cell lysates were incubated with His tagged - rRv2626c or rVector and then IP with His - agarose beads . Binding partners were confirmed by silver staining and mass spectrometric analysis . The red - colored letters indicate the peptides identified from mass spectrometry analysis .

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(A) Identification of TLR2, IRAK1, RIP1, TRAF6, MAPK11 and TOLLIP as endogenous binding partners of rRv2626c. THP-1 cell lysates were incubated with His tagged-rRv2626c or rVector and then IP with His-agarose beads. Binding partners were confirmed by silver staining and mass spectrometric analysis. The red-colored letters indicate the peptides identified from mass spectrometry analysis.


( B ) 293T cells were co - transfected with Flag - TRAF6 and Myc - Rv2626c and IP with αFlag or αMyc . WCLs were used for IB with αFlag , αMyc or αActin .

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(B) 293T cells were co-transfected with Flag-TRAF6 and Myc-Rv2626c and IP with αFlag or αMyc. WCLs were used for IB with αFlag, αMyc or αActin.


( C ) THP - 1 and BMDMs were stimulated with rRv2626c ( 2 . 5 μg / ml ) for the indicated times , followed by IP with αHis ( up ) or αTRAF6 ( down ) and IB with αTRAF6 , αHis , and αActin .

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(C) THP-1 and BMDMs were stimulated with rRv2626c (2.5 μg/ml) for the indicated times, followed by IP with αHis (up) or αTRAF6 (down) and IB with αTRAF6, αHis, and αActin.


( D ) Immunostaining of BMDMs treated with 2 . 5 μg / ml rRv2626c for 30 min . and then immunolabeled with antibody to His - rRv2626c ( Alexa Fluor 488 ) or TRAF6 ( Alexa Fluor 568 ) . DAPI ( blue ) stained nuclei .

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(D) Immunostaining of BMDMs treated with 2.5 μg/ml rRv2626c for 30 min. and then immunolabeled with antibody to His-rRv2626c (Alexa Fluor 488) or TRAF6 (Alexa Fluor 568). DAPI (blue) stained nuclei.


( A ) Schematic diagram of the structures of Rv2626c ( up ) . 293T cells were co - transfected with Flag - TRAF6 together with GST - vector or GST - Rv2626c and its mutant constructs , subjected to GST pulldown , followed by IB with αFlag . WCLs were used for IB with αGST , αFlag or αActin ( down ) .

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(A) Schematic diagram of the structures of Rv2626c (up). 293T cells were co-transfected with Flag-TRAF6 together with GST-vector or GST-Rv2626c and its mutant constructs, subjected to GST pulldown, followed by IB with αFlag. WCLs were used for IB with αGST, αFlag or αActin (down).


( B ) At 12 hr post - transfection with mammalian Myc - Rv2626c constructs together with Flag - TRAF6 and 293T cells treated with several Tat - CBS2 peptide for 12 h ( 10 µM ) for 6h , subjected to IP with αMyc , followed by IB with αFlag . WCLs were used for IB with αMyc , αFlag or αActin .

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(B) At 12 hr post-transfection with mammalian Myc-Rv2626c constructs together with Flag-TRAF6 and 293T cells treated with several Tat-CBS2 peptide for 12 h (10 µM) for 6h, subjected to IP with αMyc, followed by IB with αFlag. WCLs were used for IB with αMyc, αFlag or αActin.


( C ) Schematic diagram of the structures of TRAF6 ( up ) . 293T cells were co - transfected with Myc - Rv2626c together with GST - vector or GST - TRAF6 and its mutant constructs , subjected to GST pulldown , followed by IB with αMyc . WCLs were used for IB with αGST , αMyc or αActin ( down ) .

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(C) Schematic diagram of the structures of TRAF6 (up). 293T cells were co-transfected with Myc-Rv2626c together with GST-vector or GST-TRAF6 and its mutant constructs, subjected to GST pulldown, followed by IB with αMyc. WCLs were used for IB with αGST, αMyc or αActin (down).


( D ) 293T cells were co - transfected with Flag - TRAF6 , Myc - Rv2626c as indicated doses and HA - ubiquitin ( left ) , HA - K48 - linked ubiquitin ( middle ) , HA - K63 - linked ubiquitin ( right ) and then IP with αFlag , followed by IB with αHA . WCLs were used for IB with αFlag , αMyc , or αActin .

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(D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right) and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag , αMyc, or αActin.


( E ) BMDMs were pretreated with 2 . 5 μg / ml rRv2626c for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αTRAF6 , IB with ubiquitin , K48 - linked ubiquitin , or K63 - linked ubiquitin . WCLs were used for IB with αHis , αTRAF6 , or αActin .

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(E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin.


( B ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) .

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(B) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).


( C ) BMDMs were incubated with rRv2626c - CA , rRv2626c - DN , or rVehicle for the indicated times and then cell viability measured with MTT assay .

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(C) BMDMs were incubated with rRv2626c-CA, rRv2626c-DN, or rVehicle for the indicated times and then cell viability measured with MTT assay.


( D ) BMDMs were pretreated with rRv2626c - WT , rRv2626c - CA , or rRv2626c - DN for 1 h , and stimulated with 100 ng / ml LPS for 18 h . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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(D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( E ) Myc - Rv2626c - expressing Raw264 . 7 or THP - 1 cells were pretreated with rRv2626c - WT ( 2 . 5 μg / ml ) , rRv2626c - CA ( 10 ng / ml ) , rRv2626c - DN ( 2 . 5 μg / ml ) , for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αMyc or αTRAF6 , IB with αMyc and ubiquitin . WCLs were used for IB with αMyc , αTRAF6 , αHis or αActin .

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(E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin.


( A - C ) Schematic of the CLP model treated with rRv2626c - CA or its mutants ( upper ) . The survival of mice was monitored for 10 days ; mortality was measured for n = 25 mice per group ( lower ) . Statistical differences compared with the rVector - treated mice are indicated ( log - rank test ) .

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(A-C) Schematic of the CLP model treated with rRv2626c-CA or its mutants (upper). The survival of mice was monitored for 10 days; mortality was measured for n=25 mice per group (lower). Statistical differences compared with the rVector-treated mice are indicated (log-rank test).


( E ) representative hematoxylin and eosin ( H & E ) staining of the lung , liver and spleen ( left ) from 10 mice per group . Histopathology scores were obtained from H & E stained were determined at 20 h in CLP mice were treated with rRv2626c - CA or its mutants . Scale bar , 100 μm .

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(E) representative hematoxylin and eosin (H&E) staining of the lung, liver and spleen (left) from 10 mice per group. Histopathology scores were obtained from H&E stained were determined at 20 h in CLP mice were treated with rRv2626c-CA or its mutants. Scale bar, 100 μm.


( F ) Splenocytes were used for IP with αHis or αTRAF6 , followed by IB with αHis or αUb . WCLs were used for IB with αTRAF6 , αHis or αActin .

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(F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin.


( G ) In vivo imaging using IVIS spectrum - chromatography CT system . Intraperitoneal administration of rRv2626c - CA / Cy5 . 5 was carried out at intervals of 0 , 6 , 12 , and 18 h in a CLP - induced sepsis model . Pharmacokinetics and biodistribution were observed in spleen , liver , lung , and kidney tissues .

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(G) In vivo imaging using IVIS spectrum-chromatography CT system. Intraperitoneal administration of rRv2626c-CA/Cy5.5 was carried out at intervals of 0, 6, 12, and 18 h in a CLP-induced sepsis model. Pharmacokinetics and biodistribution were observed in spleen, liver, lung, and kidney tissues.


( H ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His were found in the spleen , liver and lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis .

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(H) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His were found in the spleen, liver and lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.


( A ) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c - CA or its mutants ( n = 10 mice per group ) .

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(A) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c-CA or its mutants (n=10 mice per group).


( B ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His or its mutants were found in the spleen or lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis .

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(B) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His or its mutants were found in the spleen or lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.


( C ) CD86 , iNOS , CD163 and Arg1 mRNA levels were determined by real - time PCR .

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(C) CD86, iNOS, CD163 and Arg1 mRNA levels were determined by real-time PCR.


( D ) Expression of SR - A , FcR , TLR2 , TLR4 , NRP1 or CXCR2 were measured by flow cytometry analysis .

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(D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.


A . Significantly recurrently mutated genes identified by MutSigCV analysis based on whole - exome sequencing data of 25 STAD patients ' samples .

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A. Significantly recurrently mutated genes identified by MutSigCV analysis based on whole-exome sequencing data of 25 STAD patients' samples.


B . Distribution of PWWP2B mutations identified in 25 STAD patients . Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset ( Network , 2014 ) . Distribution of PWWP2B mutations identified in a large - scale colorectal adenocarcinoma genomic study .

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B. Distribution of PWWP2B mutations identified in 25 STAD patients. Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset (Network, 2014). Distribution of PWWP2B mutations identified in a large-scale colorectal adenocarcinoma genomic study.


E . Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations .

E. Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations.


B . The interaction between exogenous PWWP2B and UHRF1 . The indicated plasmids were transfected into 293T cells . After 24 hours , the transfected cell lysates were immunoprecipitated ( IP ) using an anti - Myc antibody and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

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B. The interaction between exogenous PWWP2B and UHRF1. The indicated plasmids were transfected into 293T cells. After 24 hours, the transfected cell lysates were immunoprecipitated (IP) using an anti-Myc antibody and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


C . The interaction between endogenous PWWP2B and UHRF1 . Immunoprecipitation reactions were performed using rabbit IgG or anti - PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

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C. The interaction between endogenous PWWP2B and UHRF1. Immunoprecipitation reactions were performed using rabbit IgG or anti-PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


E . Myc - UHRF1 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with the anti - Myc antibody and then immunoblotted with the indicated antibodies .

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E. Myc-UHRF1 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with the anti-Myc antibody and then immunoblotted with the indicated antibodies.


G . SFB - PWWP2B WT and either Myc - UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti - FLAG and then immunoblotted with the indicated antibodies .

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G. SFB-PWWP2B WT and either Myc-UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti-FLAG and then immunoblotted with the indicated antibodies.


I Cell lysates of 293T cells transfected with Myc - UHRF1 expression vector were incubated with 2 of purified GST , GST - PWWP2B - N , or GST - PWWP2B - C recombinant proteins for 1 h at 4 ° C . After pull - down with GST - beads , the bound complexes were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) and subjected to Western blot analysis using the indicated antibodies ( upper panel ) . The Coomassie blue stain shows the purified GST fusion proteins .

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I Cell lysates of 293T cells transfected with Myc-UHRF1 expression vector were incubated with 2 of purified GST, GST-PWWP2B-N, or GST-PWWP2B-C recombinant proteins for 1 h at 4 °C. After pull-down with GST-beads, the bound complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using the indicated antibodies (upper panel). The Coomassie blue stain shows the purified GST fusion proteins.


Cell lysates of 293T expressing Myc - UHRF1 WT or Myc - UHRF1 - D4 mutants were incubated with 2 ug of purified GST or GST - PWWP2B - N for 1 h at 4 ° C . After pull - down with GST - beads , the bound complex was analyzed by Western blots .

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Cell lysates of 293T expressing Myc-UHRF1 WT or Myc-UHRF1-D4 mutants were incubated with 2 ug of purified GST or GST-PWWP2B-N for 1 h at 4 °C. After pull-down with GST-beads, the bound complex was analyzed by Western blots.


E . Comparison of the translocation kinetics for GFP - UHRF1 and mCherry - PWWP2B to the sites of DNA damage . HeLa cells expressing both GFP - UHRF1 and mCherry - PWWP2B were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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E. Comparison of the translocation kinetics for GFP-UHRF1 and mCherry-PWWP2B to the sites of DNA damage. HeLa cells expressing both GFP-UHRF1 and mCherry-PWWP2B were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


F . HeLa cells were transfected with GFP - PWWP2B WT or deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( top panel ) . The cells with positive GFP expression on the laser stripes are presented in the bar graph ( bottom panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation .

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F. HeLa cells were transfected with GFP-PWWP2B WT or deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (top panel). The cells with positive GFP expression on the laser stripes are presented in the bar graph (bottom panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation.


H . GFP - PWWP2B - A2A3 deletion mutant translocates to the DNA damage sites . HeLa cells were transfected with GFP - tagged deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( left panel ) . The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation for the cells transfected with each expression plasmid .

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H. GFP-PWWP2B-A2A3 deletion mutant translocates to the DNA damage sites. HeLa cells were transfected with GFP-tagged deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (left panel). The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation for the cells transfected with each expression plasmid.


I - J . HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP - PWWP2B - A2A3 ( I ) and GFP - PWWP2B WT ( J ) expression plasmid . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points . The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel .

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I-J. HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP-PWWP2B-A2A3 (I) and GFP-PWWP2B WT (J) expression plasmid. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points. The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel.


The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel . K . HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP - UHRF1 expression plasmids . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel. K. HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP-UHRF1 expression plasmids. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


D - G . Quantification of radiation - induced RAD51 , BRCA1 , and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs . All data were obtained from over cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 , BRCA1 , and 53BP1 foci formation . HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( D and E ) , BRCA1 ( F ) , or 53BP1 ( G ) antibodies . The cells in S phase were identified by Cyclin A staining . ( E ) The siPWWP2B - resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA - mediated PWWP2B depletion . IR - induced RAD51 foci formation was examined by immunofluorescence .

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D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.


A . Immunoblot analysis of RAD51 , BRCA1 , or 53BP1 protein levels in PWWP2B - depleted HeLa cells .

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A. Immunoblot analysis of RAD51, BRCA1, or 53BP1 protein levels in PWWP2B-depleted HeLa cells.


B . RPA phosphorylation at the indicated time points in response to IR exposure . HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4 / S8 phosphorylation and total RPA .

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B. RPA phosphorylation at the indicated time points in response to IR exposure. HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4/S8 phosphorylation and total RPA.


C - D . Quantification of radiation - induced RPA2 or S4 / S8 phosphorylated RPA foci formation in PWWP2B - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RPA2 or S4 / S8 phosphorylated RPA2 foci formation . HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RPA2 ( C ) , or S4 / S8 phosphorylation - specific RPA2 ( D ) antibodies ( bottom panel ) .

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C-D. Quantification of radiation-induced RPA2 or S4/S8 phosphorylated RPA foci formation in PWWP2B-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RPA2 or S4/S8 phosphorylated RPA2 foci formation. HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RPA2 (C), or S4/S8 phosphorylation-specific RPA2 (D) antibodies (bottom panel).


A . Immunofluorescence analysis for irradiation - induced MRE11 foci formation ( left panel ) . HeLa cells transfected with either control siRNA , siPWWP2B or siUHRF1 were irradiated , fixed and subjected to staining with MRE11 and γH2AX antibodies . More than 100 nuclei per condition were counted . The percentage of MRE11 - positive cells is presented as a bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate standard deviation .

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A. Immunofluorescence analysis for irradiation-induced MRE11 foci formation (left panel). HeLa cells transfected with either control siRNA, siPWWP2B or siUHRF1 were irradiated, fixed and subjected to staining with MRE11 and γH2AX antibodies. More than 100 nuclei per condition were counted.The percentage of MRE11-positive cells is presented as a bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate standard deviation.


B . HeLa cells expressing GFP - MRE11 were treated with control siRNA or siRNAs against PWWP2B . After 48 h , the cells were subjected to laser microirradiation . Stripes on the laser were examined at the indicated time points ( upper panel ) . The intensity of the laser stripes at the time points was determined . The average intensity values from 10 cells were graphed in the bottom panel .

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B. HeLa cells expressing GFP-MRE11 were treated with control siRNA or siRNAs against PWWP2B. After 48 h, the cells were subjected to laser microirradiation. Stripes on the laser were examined at the indicated time points (upper panel).The intensity of the laser stripes at the time points was determined. The average intensity values from 10 cells were graphed in the bottom panel.


C . The interaction between PWWP2B and MRE11 . HA - PWWP2B and Myc - MRE11 - expressing vectors were transfected into 293T cells . After 24 h , the cell lysates were immunoprecipitated with anti - HA antibody , followed by immunoblot analysis with the indicated antibodies .

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C. The interaction between PWWP2B and MRE11. HA-PWWP2B and Myc-MRE11-expressing vectors were transfected into 293T cells. After 24 h, the cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblot analysis with the indicated antibodies.


D . The interaction between endogenous PWWP2B and MRE11 . Immunoprecipitation reactions were performed using rabbit IgG or anti - MRE11 antibodies , followed by immunoblot analysis with the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

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D. The interaction between endogenous PWWP2B and MRE11. Immunoprecipitation reactions were performed using rabbit IgG or anti-MRE11 antibodies, followed by immunoblot analysis with the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


E . HA - MRE11 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with anti - HA antibody and then immunoblotted with the indicated antibodies .

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E. HA-MRE11 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with the indicated antibodies.


B - C . Comparison of IR - induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs . Quantification of radiation - induced RAD51 or BRCA1 foci formation in PWWP2B alone , UHRF1 alone or PWWP2B and UHRF1 - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 or BRCA1 foci formation . HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( B ) , or BRCA1 ( C ) antibodies ( bottom panel ) .

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B-C. Comparison of IR-induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs. Quantification of radiation-induced RAD51 or BRCA1 foci formation in PWWP2B alone, UHRF1 alone or PWWP2B and UHRF1-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RAD51 or BRCA1 foci formation. HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (B), or BRCA1 (C) antibodies (bottom panel).


B . Characterization of the various B cell progenitor fractions as described by [ 38 ] in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls .

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B. Characterization of the various B cell progenitor fractions as described by [38] in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


C . Characterization of the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls .

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C. Characterization of the indicated thymic T cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


D . Characterization of the indicated immature and mature splenic B cell populations in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls .

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D. Characterization of the indicated immature and mature splenic B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


E . Characterization of the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2 − / − littermate controls .

E. Characterization of the indicated peritoneal B cell populations in the bone marrow of 4 wildtype and 4 Shld2−/− littermate controls.


F . Left panel : Schematic of the A70 . 2 INV - 4 cell line strategy to induce RAG1 / 2 - mediated recombination using imatinib . Right panel : A70 . 2 INV - 4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1 , Shld1 , Shld2 , Shld3 , and Lig4 genes . Guide RNA targeting chicken AID was used as a negative control ( Ctrl ) . Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured ( mean ± SD of 3 biological replicates ) . The insertion - deletion ( indel ) penetrance as measured by TIDE analysis [ 39 ] of sequence for each of these sgRNA constructs is shown in Figure EV1E , and the baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F . sgRNA sequences used are shown in Table EV2 .

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F. Left panel: Schematic of the A70.2 INV-4 cell line strategy to induce RAG1/2-mediated recombination using imatinib. Right panel: A70.2 INV-4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. Guide RNA targeting chicken AID was used as a negative control (Ctrl). Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured (mean ± SD of 3 biological replicates). The insertion-deletion (indel) penetrance as measured by TIDE analysis [39] of sequence for each of these sgRNA constructs is shown in Figure EV1E, and the baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F. sgRNA sequences used are shown in Table EV2.


A . Concentration of the various indicated isotypes in the serum of 6 - 8 week old unimmunized WT and Shld2 − / − mice . Values are mean concentration ± SD of 4 biological replicates ; * * P ≤ 0 . 01 , unpaired two - tailed t - test .

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A. Concentration of the various indicated isotypes in the serum of 6-8 week old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; ** P ≤ 0.01, unpaired two-tailed t-test.


B . B cells were purified from spleens from WT and Shld2 − / − mice , and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [ 33 ] . Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported . Values are mean frequency ± SD of 4 biological replicates ; * * * * P ≤ 0 . 0001 , unpaired two - tailed t - test .

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B. B cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [33]. Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported. Values are mean frequency ± SD of 4 biological replicates; **** P ≤ 0.0001, unpaired two-tailed t-test.


C . WT , 53bp1 − / − , and Shld2 − / − mice were immunized with NP - CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP - specific antibodies of the indicated isotypes . Values are mean absorbance ± SD of 4 biological replicates .

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C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.


D . WT , 53bp1 − / − , and Shld2 − / − mice were immunized with NP - CGG , spleens were isolated and anti - IgG secreting cells were enumerated by the ELISPOT assay . Values are mean frequency ± SD of 4 biological replicates , except for Shld2 - / - ( n = 3 ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , unpaired two - tailed t test .

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D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.


A . WT and Shld2 − / − B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3 , 6 , and 9 days post - stimulation by flow cytometry . Representative plots are shown for both WT and Shld2 − / − B cells 6 - days post stimulation . The graph plots show proportion of IgG1 + and Iglo cells , mean ± SD from 6 biological replicates ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , two - way ANOVA with post hoc Dunnett ' s test .

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A. WT and Shld2−/− B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B cells 6-days post stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; ** P ≤ 0.01, *** P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test.


B . WT CH12 cells , as well as two each of 53BP1 - , SHLD1 - , SHLD2 - , and SHLD3 - deficient clones generated previously [ 21 ] , as well as a LIG4 - deficient CH12 clone were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry . Representative flow plots for WT and 53bp1 − / − CH12 cells are shown at day 3 post CIT stimulation . The graph plots show proportion of IgA + and Iglo CH12 cells , mean ± SD from 3 biological replicates ; * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s test . The letters below the x - axis represent the clone codes for the 53BP1 , SHLD1 , SHLD2 , and SHLD3 - deficient CH12 clones . One LIG4 - deficient CH12 clone was also used .

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B. WT CH12 cells, as well as two each of 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient clones generated previously [21], as well as a LIG4-deficient CH12 clone were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry. Representative flow plots for WT and 53bp1−/− CH12 cells are shown at day 3 post CIT stimulation. The graph plots show proportion of IgA+ and Iglo CH12 cells, mean ± SD from 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. The letters below the x-axis represent the clone codes for the 53BP1, SHLD1, SHLD2, and SHLD3-deficient CH12 clones. One LIG4-deficient CH12 clone was also used.


C . The Xlf , Ku70 , Ku80 , Xrcc4 , and Paxx genes were knocked out in CH12 cells by CRISPR , and two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates ; * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s test .

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C. The Xlf, Ku70, Ku80, Xrcc4, and Paxx genes were knocked out in CH12 cells by CRISPR, and two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.


A . WT , and two independent clones each of 53bp1 − / − , Shld2 − / − / − , and Shld3 − / − CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression . The Iglo population were reduced after 7 days in culture . Values are mean ± SD from 3 biological replicates ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , two - way ANOVA with post hoc Dunnett ' s test .

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A. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population were reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.


B . WT , and two independent clones each of 53bp1 − / − , Shld2 − / − / − , and Shld3 − / − CH12 clones were stimulated with CIT for 3 days . The IgM + , IgA + , and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort ( see Figure EV4B ) . Shown on bar graphs are sorted IgM + , IgA + , and Iglo populations ( each column , 1 technical replicate ) from WT and mutant CH12 clones , and the percent of cells expressing IgM , IgA , or low for both isotypes ( Iglo ) after 5 days of culture post - sort . A representative flow plot of 53bp1 − / − CH12 3 days post CIT treatment is shown .

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B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown.


A . mRNA expression analysis of IgM and IgA was carried out by RT - PCR for Iglo subclones from WT and two independent 53bp1 − / − CH12 ( TA and T1 ) clones . The Iglo subclones derived from the 53bp1 − / − CH12 TA clone are listed as D01 - D18 , while the Iglo subclones derived from the 53bp1 − / − CH12 T1 clone are listed as F03 - F24 . Gels show the RT - PCR analysis for IgM cDNA ( top ) , IgA cDNA ( middle ) , and AID cDNA ( bottom ) as control .

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A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control.


B . mRNA expression analysis as in A of Iglo subclones of two independent Shld2 - / - / - ( F2 and F3 ) clones , and IgA + subclones from Shld2 - / - / - clone F3 . The Iglo subclones derived from the Shld2 - / - / - CH12 F2 clone are listed as H03 - H40 , while the Iglo subclones derived from the Shld2 - / - / - CH12 F3 clone are listed as J02 - J109 .

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B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109.


Long - range PCR of Iglo subclones from WT and two independent 53bp1 - / - CH12 ( TA and T1 ) clones . The Iglo subclones derived from the 53bp1 − / − CH12 TA clone are listed as D01 - D18 , while the Iglo subclones derived from the 53bp1 − / − CH12 T1 clone are listed as F03 - F24 . Gels show long - range amplicons ( top ) and AID genomic DNA PCR control ( bottom ) .

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Long-range PCR of Iglo subclones from WT and two independent 53bp1-/- CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show long-range amplicons (top) and AID genomic DNA PCR control (bottom).


Long - range PCR as in B of Iglo subclones of two independent Shld2 - / - / - ( F2 and F3 ) clones ( left 2 panels ) , and IgA + subclones from Shld2 - / - / - clone F3 .

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Long-range PCR as in B of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3.


Sequence analysis of a representative subset of long range - PCR amplicons . Symbol annotations : ( * ) inverted sequence , ( ◊ ) rearranged fragment order , ( † ) amplified with the EμF primer rather than IμF , ( ‡ ) undamaged Cα coding sequence .

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Sequence analysis of a representative subset of long range-PCR amplicons. Symbol annotations: (*) inverted sequence, (◊) rearranged fragment order, (†) amplified with the EμF primer rather than IμF, (‡) undamaged Cα coding sequence.


( A ) Volcano plot representation of PARL interaction partners . Mitochondria isolated from PARL - / - FITR293Tcells expressing PARL - FLAG were subjected to co - immunoprecipitation ( IP ) . Co - purifying proteins were separated by SDS - PAGE and identified by quantitative MS ( n = 3 ) ( Dataset EV1 ) .

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(A) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293Tcells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1).


( B ) Heat map of Log2 transformed LFQ intensities for three independent experiments . All significant ( FDR < 0 . 05 ) proteins showing a positive ratio between PARLIP and control in ( A ) are shown . Grey color shows missing quantitative information . Clustering was performed using the complete method with Euclidean distance .

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(B) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARLIP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.


( C ) Complexome analysis of MEFmitochondria ( n = 3 ) . Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0 . 05 ( A ) and identified by mass spectrometry after BN - PAGE analysis are shown after hierarchical clustering .

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(C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.


( D ) BN - PAGE analysis of mitochondria lacking SLP2 , YME1L or PARL . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 13 % blue native gradient gel and analyzed using SLP2 - , YME1L - and PARL - specific antibodies .

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(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.


( E ) High resolution complexome analysis of SPY complex members SLP2 , YME1L and PARL using MEFmitochondria ( n = 3 ) . Heat maps and migration profiles are shown after separation using a 3 - 9 % BN - PAGE . SLP2 , YME1L and PARL migrate in a high molecular weight complex ( 2 MDa ) , whereas SLP2 additionally is present in complexes of 1 . 6 MDa ) .

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(E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).


( A ) Co - immunoprecipitation of endogenous SLP2 , PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2 - or PARL - specific antibodies . IgG was used as a negative control . In , input ( 10 % ) .

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(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).


( B , C ) Co - immunoprecipitation of endogenous SLP2 and YME1L with PARL - specific antibodies in mitochondria isolated from wild type MEFs ( WT ) and MEFs lacking YME1L , SLP2 or PARL . In , input ( 10 % ) .

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(B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).


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( D ) Assembly of SLP2 , PARL and YME1L into a high molecular weight complex . Mitochondria isolated from FITR293T cells ( Con ) and cells inducibly expressing SLP2 - FLAG ( FLAG ) were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , PARL - , YME1L - and PHB2 - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) .

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(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).


( E ) High resolution BN - PAGE analysis of mitochondria lacking PARL , YME1L or SLP2 . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 9 % gradient gel containing 0 - 10 % glycerol and analyzed using SLP2 - specific antibodies .

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(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.


( F , G ) Submitochondrial localization of SLP2 . ( F ) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated . Fractions were analyzed by SDS - PAGE and immunoblotting . MFN2 served as OM marker , TIMM23 , SMAC / DIABLO , and YME1L as IMS markers , and AFG3L1 and AFG3L2 as matrix marker proteins .

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(F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.


( F , G ) Submitochondrial localization of SLP2 . ( G ) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet ( P ) and supernatant ( S ) fraction by centrifugation . Fractions were analyzed by SDS - PAGE and immunoblotting .

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(F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.


( A ) Steady state levels of proteolytic substrates of YME1L . Whole cell extracts of Yme1l - / - , Parl - / - , and Slp2 - / - MEFs were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies .

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(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.


( B ) PGAM5 processing depends on proteolytically active PARL . PARL and catalytic inactive PARLS277A harboring C - terminal FLAG - epitopes were expressed under the control of a tetracycline ( tet ) - inducible promoter in wild type ( WT ) and PARL - / - FITR293T cells as indicated . Processing of L - PGAM5 to S - PGAM5 was monitored by immunoblotting .

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(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.


( C ) Accelerated processing of L - PGAM5 in Slp2 - / - cells . Processing of L - PGAM5 was analyzed by immunoblotting in wild type ( WT ) , Yme1l - / - , Parl - / - and Slp2 - / - MEFs after inhibition of cytosolic protein synthesis by cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown in the lower panel . Two - way ANOVA analysis ( n = 3 ; * * * * , p < 0 . 0001 ) .

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(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).


( D ) The accelerated processing of L - PGAM5 in Slp2 - / - cells is mediated by PARL . PARL was depleted from Slp2 - / - cells by RNAi prior to CHX treatment . A quantification of L - PGAM5 levels at different time points is shown . Two - way ANOVA analysis ( n = 3 ; * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) .

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(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).


( E ) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin were analyzed by BN - PAGE and immunoblotting using FLAG - and PGAM5 - specific antibodies .

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(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.


( A ) PARL dependent cleavage of PINK1 - HA is reduced in Slp2 - / - and Yme1l - / - MEFs . Whole cell extracts of Slp2 - / - , Parl - / - and Yme1l - / - MEFs expressing PINK1 - HA were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h . ( B ) Quantification of the protein ratio ( Log2 ) PINK1 - HA 52 kDa / 63 kDa ( n = 3 ; * , p < 0 . 05 ; One - way ANOVA ) . n . s . , not significant . Error bars indicate SEM .

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(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.


( C ) PINK1 - HA associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using FLAG - , HA - and SLP2 - specific antibodies .

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(C) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.


( D ) PINK1 associates with SLP2 . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells , depleted of SLP2 by siRNA as indicated , were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using SLP2 - and PINK1 - specific antibodies .

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(D) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.


( A ) Processing of L - PGAM5 to S - PGAM5 in depolarized mitochondria of wild type ( WT ) , Slp2 - / - , Yme11 - / - , and Parl - / - MEFs . PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown . L - PGAM5 levels at t = 0 was set to 100 % . Two - way ANOVA analysis ( n = 3 ; * , p < 0 . 05 , * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) . Arrowheads denote intermediate PGAM5 cleavage products .

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(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.


( B ) OMA1 - mediated processing of PGAM5 in depolarized Parl - / - mitochondria . Processing of L - PGAM5 to S - PGAM5 in Parl - / mitochondria was assessed as in ( A ) in the presence or absence of CCCP ( 20 µM ) , which were depleted of OMA1 by siRNA as indicated . The arrowhead denotes an intermediate PGAM5 cleavage product . SCR , scrambled .

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(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.


( C ) Impaired PGAM5 processing in depolarized Oma1 - / - mitochondria lacking PARL . Processing of L - PGAM5 to S - PGAM5 was examined as in ( A ) after 2 h in wild type ( WT ) and Oma1 - / - mitochondria in the presence or absence of CCCP ( 20 µM , 2 h ) , which were depleted of PARL by siRNA as indicated . * unspecific cross - reaction .

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

(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.


( D ) OMA1 mediates accelerated processing of L - OPA1 in Slp2 - / - MEFs . Processing of OPA1 and PGAM5 was monitored in Slp2 - / - , Oma1 - / - , and Slp2 - / - Oma1 - / - cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) .

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

(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).


( E ) OMA1 interacts with SLP2 . OMA1 - / - FITR293T cells ectopically expressing OMA1 - myc were transfected with pcDNA5 ( Control ) or pcDNA5 - SLP2 - FLAG ( FLAG ) . Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , YME1L - , PARL - , and myc - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) . Samples were analyzed by SDS - PAGE in parallel ( lower panel ) . ( F ) Quantification of immunoprecipitation efficiencies in ( E ) .

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

(E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).


B , C , D . WT , ∆ cpt1 ∆ ept1 ( B ) , Δcho2 ( C ) or Δopi3 ( D ) cells expressing GFP - Atg8 were grown to logarithmic phase in SD - URA , and shifted to SD - N for 4 h of starvation . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was used as a loading control . Middle panel - Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by student ' s t - test ( paired , two tailed ) ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount , during SD and SD - N .

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

B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N.


E . WT , Δopi3 , and Δcho2 as well as Δatg1 ( negative autophagy control ) cells expressing chromosomally - tagged Fba1 - GFP were grown to log phase in SD , and starved in SD - N for 4 hours . Cells were harvested at indicated starvation time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Right panel - Autophagic activity during starvation was quantified , by calculating the ratio of free GFP to total GFP ( Fba1 - GFP + free GFP ) .

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

E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP).


F . WT , Δopi3 , Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain , were grown to log phase in SD , and starved in SD - N for 4 hours . Pho8 activity was measured by the alkaline phosphatase 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 B-MEASURED_VAR O O O O O O O O O

F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay.


A . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT , Δopi3 and Δcho2 cells expressing GFP - Atg8 were grown to log phase in SD - URA medium and shifted to SD - N for 3 h . Cells were observed by confocal microscopy before ( SD ) and during nitrogen starvation ( SD - N ) ( left panel ) . Scale bar 1 µm . Right panel - Quantification of of cells with or without GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 ) ( ns - not significant ) , error bars represent SEM of at least 3 independent experiments . Number of cells analyzed for each strain and condition : SD ( WT ( n = 446 ) , Δcho2 ( n = 272 ) , Δopi3 ( n = 272 ) ) , SD - N ( WT ( n = 398 ) , Δcho2 ( n = 224 ) , Δopi3 ( n = 213 ) ) .

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

A. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).


A , B . WT and Δopi3 cells expressing GFP - Atg8 were grown to log phase in SD - URA , and shifted to SD - N for 4 h , choline ( 1 mM ) was not supplemented ( - choline ) or supplemented to SD and SD - N ( + choline SD , SD - N ) or only to SD - N ( + choline , SD - N ) as indicated . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Middle panel - Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth ( SD ) or during starvation ( SD - N ) , with choline supplemented as indicated . Statistical analysis was done by Sidak ' s multiple comparisons test compared to WT ( ns - not significant , * , p ≤ 0 . 05 , * * , p ≤ 0 . 005 , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments .

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

A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.


C . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT and Δopi3 cells expressing GFP - Atg8 were grown in SD - URA and shifted to SD - N . Choline ( 1mM ) was either excluded ( - choline ) , added to growth and starvation medium ( + choline SD , SD - N ) or added only to starvation medium ( + choline , SD - N ) . Cells were observed using confocal microscopy during starvation ( left panel ) . Scale bar 1 µm . Right panel - Quantification of cells with GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , error bars represent SEM of 3 independent experiments . Number of cells analyzed for each strain condition ; - Choline : WT ( n = 487 ) , Δopi3 ( n = 316 ) , Choline SD , SD - N : WT ( n = 251 ) , Δopi3 ( n = 361 ) , Choline SD - N : WT ( n = 320 ) , Δopi3 ( n = 267 ) . D . PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP - Atg8 , with or without choline ( 1 mM ) supplementation to SD - N . Samples were taken after 4 h in SD - N medium .

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

C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.


A . Representative Images of Atg5 - mNG with sfTq2 - Atg8 during SD - N . Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium , shifted to SD - N for 4 h , and observed by Widefield microscopy ( left panel ) , scale bar 1 µm . Right panel - quantification of percentage of cells with Atg5 puncta during different time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ns - not significant ) , error bars represent SEM of 2 independent experiments . Number of cells analyzed for each strain and time point ( 0 hour : Δatg3 ( n = 296 ) , Δatg3Δopi3 ( n = 111 ) , 1 hour : Δatg3 ( n = 325 ) , Δatg3Δopi3 ( n = 110 ) , 2 hours : Δatg3 ( n = 338 ) , Δatg3Δopi3 ( n = 135 ) , 4 hours : Δatg3 ( n = 309 ) , Δatg3Δopi3 ( n = 167 ) .

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

A. Representative Images of Atg5-mNG with sfTq2-Atg8 during SD-N. Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium, shifted to SD-N for 4 h, and observed by Widefield microscopy (left panel), scale bar 1 µm. Right panel- quantification of percentage of cells with Atg5 puncta during different time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's ns- not significant), error bars represent SEM of 2 independent experiments. Number of cells analyzed for each strain and time point (0 hour: Δatg3 (n=296), Δatg3Δopi3 (n=111), 1 hour: Δatg3 (n=325), Δatg3Δopi3 (n=110), 2 hours: Δatg3 (n=338), Δatg3Δopi3 (n=135), 4 hours: Δatg3 (n=309), Δatg3Δopi3 (n=167).


C . Representative images of WT and Δopi3 cells expressing GFP - Atg8 . Cells were grown to log phase in SD - URA medium , shifted to SD - N for 3 h and observed by Airyscan microscopy ( left panel ) . White dashes indicate cell boundaries , yellow dashes indicate phagophores . For each cell - Magnification of yellow dashed area ( top right ) , schematic representation ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 structures imaged by Airyscan microscopy . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * p ≤ 0 . 05 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 45 ) , Δopi3 ( n = 110 ) .

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

C. Representative images of WT and Δopi3 cells expressing GFP-Atg8. Cells were grown to log phase in SD-URA medium, shifted to SD-N for 3 h and observed by Airyscan microscopy (left panel). White dashes indicate cell boundaries, yellow dashes indicate phagophores. For each cell - Magnification of yellow dashed area (top right), schematic representation (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 structures imaged by Airyscan microscopy. Statistical analysis was done by student's t-test (unpaired) (** p≤0.05), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=45), Δopi3 (n=110).


D . Representative CLEM images of WT and Δopi3 cells expressing GFP - Atg8 , grown to log phase in SD - URA medium and shifted to SD - N for 3h . Cells were harvested , deep frozen and processed for CLEM ( left panel ) , V - vacuole . For Δopi3 cell - Magnification of yellow dashed area of GFP - Atg8 positive phagophore ( top right ) , TEM image only ( middle right ) , schematic representation of a phagophore ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 positive cup shaped membrane structures imaged by CLEM . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * * p ≤ 0 . 005 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 20 ) , Δopi3 ( n = 21 ) .

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

D. Representative CLEM images of WT and Δopi3 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 3h. Cells were harvested, deep frozen and processed for CLEM (left panel), V-vacuole. For Δopi3 cell - Magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of a phagophore (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 positive cup shaped membrane structures imaged by CLEM. Statistical analysis was done by student's t-test (unpaired) (*** p≤0.005), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=20), Δopi3 (n=21).


A . Representative images of Δypt7 , Δopi3Δypt7 expressing GFP - Atg8 ( z - stack projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , images were taken during starvation in indicated time points by widefield microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of GFP - Atg8 puncta per cell in Δypt7 , Δopi3Δypt7 at indicated time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , compared to WT . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and time point , 0 hours : Δypt7 ( n = 322 ) , Δopi3Δypt7 ( n = 194 ) , 1 . 5 hours : Δypt7 ( n = 220 ) , Δopi3Δypt7 ( n = 155 ) , 3 . 5 hours Δypt7 ( n = 216 ) , Δopi3Δypt7 ( n = 307 ) . B . Quantification of cup - shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP - Atg8 during starvation ( 1 - 3h ) . Statistical analysis was done by students t - test ( * * * , p ≤ 0 . 001 ) , error bars represent SEM from at least 3 independent experiments . Number of cells analyzed for each strain , Δypt7 ( n = 347 ) , Δopi3Δypt7 ( n = 474 ) .

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

A. Representative images of Δypt7, Δopi3Δypt7 expressing GFP-Atg8 (z-stack projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, images were taken during starvation in indicated time points by widefield microscopy (left panel). Scale bar 1 µm. Right panel- quantification of GFP-Atg8 puncta per cell in Δypt7, Δopi3Δypt7 at indicated time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), compared to WT. Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and time point, 0 hours: Δypt7 (n=322), Δopi3Δypt7 (n=194), 1.5 hours: Δypt7 (n=220), Δopi3Δypt7 (n=155), 3.5 hours Δypt7 (n=216), Δopi3Δypt7 (n=307). B. Quantification of cup-shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP-Atg8 during starvation (1-3h). Statistical analysis was done by students t-test (***, p≤0.001), error bars represent SEM from at least 3 independent experiments. Number of cells analyzed for each strain, Δypt7 (n= 347), Δopi3Δypt7 (n= 474).


C . Representative images of mNG - tagged Atg8 colocalized with mScarletI - PXVam7 . WT , Δopi3 , Δatg1 and Δopi3Δatg1 cells on the background of Δymr1 , expressing mScarletI - PXVam7 under the CUP1 promoter from the knocked - out VPS38 locus , were grown to log phase in SD medium in the presence of 10 µM copper sulfate , and shifted to SD - N in the presence of 10 µM copper sulfate . Images were taken during SD - N ( 1 - 3 h ) by widefield microscopy . D . Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 ( C ) by ImageJ . Statistical analysis was done by student ' s t - test , unpaired , two sided ( ns - not significant ) , error bars represent SEM of at least three independent experiments . Number of cells analyzed for each strain , WT ( n = 51 ) , Δopi3 ( n = 28 ) .

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

C. Representative images of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).