Figure 1 ( A ) Cellomics images ( top ) of GFP - LC3 ‐ HEK cells in full medium ( FM ) or after 2 h in starvation medium ( ES ) with leupeptin ( EL ) . Scale bars = 40 μm . Optimised automated analysis ( bottom left ) programme detecting hoechst ‐ labelled nuclei ( purple line ) , cell outline ( red line ) and identifies GFP - LC3 spots ( red spots ) in FM and EL . Enlarged GFP - LC3 images in FM and EL ( bottom right ) . Scale bars = 10 μm . ( B ) SCPO , STIPO and STAPO after indicated siRNA treatment of GFP - LC3 ‐ HEK cells incubated as in ( A ) . Error bars represent s . e . m . Significance was determined using a two ‐ tailed paired t ‐ test compared with RISCfree ( RF ) in EL : SCPO siULK1 , * * * P = 0 . 0002 ; siATG7 , * * P = 0 . 0078 ; siNRBP2 , * * P = 0 . 0088 . STIPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0060 ; siNRBP2 , * * P = 0 . 0041 . STAPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0037 ; siNRBP2 , * * P = 0 . 0028 . The experiments were performed : RISCfree ( n = 5 ) , siULK1 ( n = 5 ) , siATG7 ( n = 3 ) , siNRBP2 ( n = 3 ) . ( D ) Scatter plot of median SCPO Z ‐ scores from siRNA pools in the primary screen that met the minimum object count ( OC ) cutoff . Dashed lines represent approximate cutoff for 500 best increasers and decreasers . RP of known autophagy genes ( ATG4C , ATG14 , RB1CC1 , ATG9A , ATG7 ) are given in Supplementary Table S2 ; AMBRA1 ( FLJ20294 ) , TBC1D25 ( OATL1 ) and SH3GLB1 ( BIF ‐ 1 ) in Supplementary Table S1 ; LAMTOR2 has a RP = 708 . 7 and RUBICON has a RP = 1126 .

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

Figure 1(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.(B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3).(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.


Figure 2 ( A ) SCPO , STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP - LC3 spot phenotype , above a cutoff of greater or less than 20 % of control , and passing additional criteria ( Supplementary Table 3 ) . Red boxes indicate an increase > 1 . 2 ; pink boxes an increase between 1 . 1 and 1 . 2 ; white boxes are between 0 . 9 and 1 . 1 ; light blue boxes a decrease between 0 . 9 and 0 . 8 ; dark blue boxes a decrease to 0 . 8 . Hits underlined were taken forward as three out of four4 hits , ( § ) not chosen because of restricted tissue expression , ( * ) are three out of four when alternate method of normalisation is used . ( B ) Table summarising results from further validation of 20 genes . mRNA levels scored as ( + ) or ( − ) indicate efficient or poor knockdown , respectively ( see Supplementary Figures S1 and S2 ) . Increasers ( left ) or decreasers ( right ) assayed for LC3 lipidation were scored ( + ) for yes , ( − ) for no , ( + / − ) subtle change and ( nd ) not done ( Supplementary Figure S3 ) . ( C - E ) STIPO results after incubation under different autophagy ‐ inducing conditions following ( C ) SCOC knockdown ( D ) SUPT5H knockdown and ( E ) WAC knockdown . siRNA ‐ treated cells were incubated for the indicated time in FL : full medium plus leupeptin ; ES ; EL : ES plus leupeptin ; Torin : 250 nM Torin1 in FL ; Resv : 128 μM Resveratrol in FL ; LiCl : 10 mM LiCl in FM .

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

Figure 2(A) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP-LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used.(B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3).(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.


Figure 3 ( B ) Endogenous SCOC shows colocalisation with Atg9 ( red ) and TGN46 ( blue ) . HEK293 cells were fixed and permeabilised in saponin . Colocalisation indicated with arrows . ( C ) Endogenous SCOC ( green top , red bottom ) partially colocalises with endogenous LC3 ( red , top ) and GFP - LC3 ( green , bottom ) in HEK293 and GFP - LC3 ‐ HEK cells , respectively , after 2 h amino ‐ acid starvation . Boxed region is at higher magnification in merge panel on right . Scale bars = 10 μm .

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

Figure 3(B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows.(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.


Figure 4 ( A ) SCOC is required for autophagy in HEK293 cells . Anti ‐ ULK1 , ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siSCOC ‐ 03 EL , * P = 0 . 0419 . ( B ) SCOC is required for p62 degradation . p62 levels were determined by western blot ( Supplementary Figure S5 ) after incubation in FM , ES for 2 or 4 h , or ES with BafilomycinA1 ( ES + BAF ) for 2 and 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree ES 2 and 4 h , * * * P > 0 . 0001 and * * 0 . 0014 , respectively . ( C ) SCOC - GFP and SCOC - myc levels are reduced by SCOC siRNA duplex ‐ 03 . Anti ‐ GFP , ‐ Myc and ‐ Actin blots of HEK293 cells after treatment with ULK1 , RISCfree or SCOC siRNA and overexpression of GFP , SCOC - myc or SCOC - GFP . ( D ) Overexpression of SCOC - myc increases autophagy . Anti ‐ Actin , ‐ LC3 and ‐ Myc blots of HEK293 cells after transfection of pcDNA or SCOC - myc . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 5 ) . SCOC - myc EL versus pcDNA EL , * P = 0 . 0215 . ( E ) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells . Arrows show partial colocalisation . Boxed region is at higher magnification in merge panel on bottom right . Scale bars = 20 μm .

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

Figure 4(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419.(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP.(D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215.(E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.


Figure 5 ( A ) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation . Anti ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells . Quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RISCfree ES versus siFEZ1 ‐ 01 ES , P = 0 . 034 . Quantification of p62 / actin ( Supplementary Figure S5 ) performed as in Figure 4B ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree 2 and 4 hP > 0 . 0001 and P = 0 . 0014 , respectively . qRT - PCR showing FEZ1 mRNA levels after indicated knockdown in GFP - LC3 ‐ HEK cells ( error bars represent s . d . ) . ( B ) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation . Anti ‐ Myc , ‐ Actin and ‐ LC3 blot after transfection with pcDNA or Myc - FEZ1 in HEK293 cells . Quantification of LC3II / actin ; error bars represent s . e . m . ( n = 3 ) ; pcDNA ES versus Myc - FEZ1 ES , P = 0 . 0318 . Anti ‐ GFP , ‐ Actin and ‐ p62 blot after transfection with GFP or FEZ1 - GFP and treatment with ES or ES plus Bafilomycin ( EB ) for 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; GFP FM versus GFP ES , P = 0 . 0077 ; GFP FM versus GFP EB , P = 0 . 0212 ; GFP ES versus FEZ1 - GFP ES , P = 0 . 048 . ( C ) SCOC and FEZ1 interact . Left panel shows anti ‐ SCOC and ‐ FEZ1 blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of SCOC - myc and FEZ1 - GFP with anti ‐ GFP antibody . Input lysate ( Inp ) , unbound supernatant ( Unb ) . Right panel shows anti ‐ FEZ1 and ‐ FLAG blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of FLAG - SCOC and Myc - FEZ1 with anti ‐ FLAG antibody . ( D ) FEZ1 interacts with SCOC in vitro . GST ‐ pulldown assays using in vitro translated 35S ‐ labelled Myc - FEZ1 ( right ) or 35S ‐ labelled Myc ‐ control ( left ) and GST or GST - SCOC ( asterisk in bottom panel ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) , protein loading by coomassie blue ( bottom ) . ( E ) The FEZ1 - SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled ‐ coil domain of FEZ1 as demonstrated in GST ‐ pulldown assays with in vitro translated 35S ‐ labelled WT or mutant Myc ‐ FEZ1 and GST or GST ‐ SCOC . Autoradiographs of 10 % input and bound proteins ( top panels ) and Coomassie blue ‐ stained gels of bead ‐ bound GST and GST ‐ SCOC proteins used ( bottom panels ) .

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

Figure 5(A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin ( Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 hP>0.0001 and P=0.0014, respectively. qRT-PCR showing FEZ1 mRNA levels after indicated knockdown in GFP-LC3‐HEK cells (error bars represent s.d.).(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048.(C) SCOC and FEZ1 interact. Left panel shows anti‐SCOC and ‐FEZ1 blots after co‐expression in HEK293 cells and co‐immunoprecipitation of SCOC-myc and FEZ1-GFP with anti‐GFP antibody. Input lysate (Inp), unbound supernatant (Unb). Right panel shows anti‐FEZ1 and ‐FLAG blots after co‐expression in HEK293 cells and co‐immunoprecipitation of FLAG-SCOC and Myc-FEZ1 with anti‐FLAG antibody.(D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc-FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST-SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS-PAGE (top), protein loading by coomassie blue (bottom).(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).


Figure 6 ( A ) FEZ1 interacts with both the kinase domain ( 1 - 278 ) and the proline - serine rich spacer of ULK1 ( 279 - 828 ) . GFP ‐ or Myc ‐ tagged ULK1 fragments were in vitro translated with 35S ‐ methionine and subjected to GST ‐ pulldown assays using GST and GST - FEZ1 purified from E . coli ( bottom ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) . ( B ) ULK1 interacts with FEZ1 . ULK1 was immunoprecipitated from HEK293 lysates with anti ‐ ULK1 and bound ULK1 and FEZ1 were detected with anti ‐ ULK1 and anti ‐ FEZ1 antibodies . Input lysate ( Inp ) . ( C ) The FEZ1 - GFP and Myc - ULK1 interaction is unaffected by amino ‐ acid starvation . Anti ‐ Myc and ‐ GFP blots after HEK293 cells were co ‐ transfected with FEZ1 - GFP and Myc - ULK1 , incubated in FM or ES for 2 h , harvested and the complex was immunoprecipitated using anti ‐ GFP antibody . ( D ) BN - PAGE reveals SCOC and FEZ1 are in a complex . FEZ1 - GFP , FLAG - SCOC and Myc - ULK1 wild type or kinase ‐ inactive ULK1K46I ( ULK1KI ) were co ‐ expressed in HEK293 cells . Anti ‐ GFP , ‐ FLAG and ‐ Myc western blots are shown for the BN - PAGE gel ( top ) or the SDS - PAGE gel ( bottom ) . In GFP and FLAG western blots , two different exposures of the same BN - PAGE blot are shown spliced together . ( E ) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC . RF or ULK1 siRNA ‐ treated cells were co ‐ transfected with FEZ1 - GFP and FLAG - SCOC , then incubated and immunoprecipitated as in ( C ) . Western blot of lysates for ULK1 , GFP and FLAG , and immunoprecipitates with GFP and FLAG antibodies . ( F ) siRNA depletion of SCOC reduces FEZ1 - ULK1 complex . HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1 - GFP . FEZ1 - GFP was detected with anti ‐ GFP in both BN - PAGE and SDS - PAGE . Endogenous ULK1 was detected with anti ‐ ULK1 antibody . Arrowheads indicate a FEZ1 - GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex > 1236 kDa also detected in ( D ) in the triple transfection . The 200 KDa complex is sensitive to loss of SCOC and ULK1 . ( G ) SCOC can reduce the interaction of ULK1 with MBP - FEZ1 . Immobilised MBP - FEZ1 was incubated with in vitro 35S ‐ methionine ‐ labelled Myc - ULK1 . Addition of increasing amounts of in vitro translated 35S ‐ methionine SCOC reduced the amount of Myc - ULK1 bound . Top and bottom panels are as described in ( A ) .

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

Figure 6(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top).(B) ULK1 interacts with FEZ1. ULK1 was immunoprecipitated from HEK293 lysates with anti‐ULK1 and bound ULK1 and FEZ1 were detected with anti‐ULK1 and anti‐FEZ1 antibodies. Input lysate (Inp).(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.(D) BN-PAGE reveals SCOC and FEZ1 are in a complex. FEZ1-GFP, FLAG-SCOC and Myc-ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN-PAGE gel (top) or the SDS-PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN-PAGE blot are shown spliced together.(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies.(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1.(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).


Figure 7 ( A ) SCOC interacts with UVRAG . HEK293 cells were transfected with FLAG - SCOC and Myc - UVRAG , lysed and incubated with anti ‐ FLAG ( left ) or anti ‐ Myc ( right ) antibodies . Co ‐ immunoprecipitation of Myc - UVRAG ( left ) and FLAG - SCOC ( right ) was confirmed using anti ‐ Myc and anti ‐ FLAG antibodies for western blotting . Asterisk in ( A - C ) and ( D ) indicates non ‐ specific bands . ( B ) Interaction of SCOC with UVRAG is unaffected by FEZ1 - GFP . As in ( A ) , co ‐ immunoprecipitation was preformed in HEK293 cells transfected with FLAG - SCOC , Myc - UVRAG and FEZ1 - GFP . Myc - UVRAG was detected as in ( A ) , FEZ1 - GFP was detected with anti ‐ GFP antibody . ( C ) The SCOC and UVRAG interaction is reduced by starvation , and this reduction is inhibited by FEZ1 - GFP . HEK293 cells were transfected as in ( B ) and 24 h later either treated with FM or ES for 2 h . FLAG - SCOC or Myc - UVRAG was immunoprecipitated with anti ‐ FLAG or anti ‐ Myc antibodies and the bound proteins were detected with anti ‐ Myc , ‐ GFP or ‐ FLAG antibodies by western blotting . Quantification of the amount of FLAG - SCOC co ‐ immunoprecipitated with Myc - UVRAG in FM or ES , without overexpressed FEZ1 - GFP ( left ) or with overexpressed FEZ1 - GFP ( right ) . Significance was determined using a two ‐ tailed paired t ‐ test ( n = 3 ) . ( D ) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction . HEK293 cells were transfected with FEZ1 - GFP , GFP or FLAG - SCOC alone or in combination . Top panel input , bottom panel immunoprecipitation with anti ‐ GFP or anti ‐ FLAG . Western blot for endogenous UVRAG , GFP - FEZ1 GFP or FLAG - SCOC as indicated . Arrowhead : endogenous UVRAG . ( E ) UVRAG can interact with both FEZ1 and SCOC in vitro . GST , GST - FEZ1 or GST - SCOC were immobilised and incubated with 35S ‐ labelled Myc - UVRAG as indicated . Arrowheads indicate GST - FEZ1 ( left ) and GST - SCOC ( right ) . ( F ) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells . Arrows show colocalisation . Scale bar = 10 μm .

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

Figure 7(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands.(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody.(C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3).(D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG.(E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right).(F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.


Figure 8 ( A ) Depletion of endogenous WAC . Anti ‐ WAC and ‐ Actin blot and WAC mRNA levels by qRT - PCR after treatment with RF or individual WAC siRNAs ( W ‐ 01 to W ‐ 04 ) in HEK293 cells . qRT - PCR error bars represent standard deviation of triplicates ( n = 2 ) . ( B ) ( B ) WAC is required for autophagy . Anti ‐ ULK1 , ‐ WAC , ‐ Actin and ‐ LC3 blots after siRNA treatment in HEK293 cells and incubation in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siWAC ‐ 03 EL , * * P = 0 . 0079 . ( C ) p62 degradation requires WAC . Anti ‐ p62 and ‐ Actin blot after siRNA treatment of GFP - LC3 ‐ HEK cells in FM or ES for 2 h in duplicate . Bars represent p62 / actin levels normalised to RF FM ; quantification of p62 / actin ; error bars represent s . e . m . ( n = 3 ) : RF FM versus RF ES , * * P = 0 . 0057 ; RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0025 ; RF ES versus siWAC ‐ 03 ES , * P = 0 . 0386 . ( D ) WAC depletion accelerates degradation of a UPS reporter . Anti ‐ p62 , ‐ GFP , ‐ Actin , ‐ LC3 , ‐ WAC and ‐ Atg16 blots after siRNA treatment in UbG76V - YFP MelJuSo cells and incubation conditions FM , ES or FM with 10 μM MG132 ( MG ) for 2 h . Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC ‐ 03 siRNA treatment ( n = 3 ) and compared with ATG16 siRNA treatment ( n = 2 ) . Error bars represent s . e . m . : RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0082 . ( E ) Soluble HttQ25 - CFP degradation is increased after WAC depletion . Anti ‐ WAC , ‐ Actin and ‐ GFP blots after indicated siRNA treatment ( siWAC duplexes ‐ 02 , ‐ 03 , ‐ 04 ) of HttPolyQ ‐ mutant HeLa cell lines and incubation conditions DMSO or DOX ( Doxycyline ) for 3 days . The insoluble fraction was obtained by pelleting the HttQ103 lysates ( see Materials and methods ) . Quantification of normalised average HttQ25 - CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment ; error bar represents s . e . m . ( n = 3 ) : HttQ25 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0350 ; soluble HttQ103 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0380 .

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

Figure 8(A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT-PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT-PCR error bars represent standard deviation of triplicates (n=2).(B) (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079.(C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP-LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386.(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082.(E) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.


Figure 1 ( C ) Representative image of mESC expressing Nanog - EGFP in + LIF . Bright field , epifluorescence , HILOM , and a max projection image of 2 , 000 frames are shown . ( F ) Confocal images of DRAQ5 staining , showing double stranded DNA ( top ) , immunostaining of H3K27ac ( middle ) , and Nanog - EGFP ( bottom ) , along with the progression of differentiation ( + 2i , + LIF , − LIF , and − LIF 2d ) . ( G ) Swarmplots and violinplots of coefficient of variation of DRAQ5 fluorescence in a nucleus of an mESC expressing Nanog - EGFP at the condition of + 2i ( red , n = 20 cells ) , + LIF ( blue , n = 20 ) , − LIF ( green , n = 23 ) , − LIF + TSA ( light green , n = 22 ) , − LIF2d ( purple , n = 21 ) , and − LIF2d + TSA ( pink , n = 19 ) . Double asterisks and single asterisks indicate p - value < 0 . 05 and 0 . 05 ≤ p - value < 0 . 1 , respectively , in Student ' s t - test .

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

Figure 1(C) Representative image of mESC expressing Nanog-EGFP in +LIF. Bright field, epifluorescence, HILOM, and a max projection image of 2,000 frames are shown.(F) Confocal images of DRAQ5 staining, showing double stranded DNA (top), immunostaining of H3K27ac (middle), and Nanog-EGFP (bottom), along with the progression of differentiation (+2i, +LIF, −LIF, and −LIF 2d).(G) Swarmplots and violinplots of coefficient of variation of DRAQ5 fluorescence in a nucleus of an mESC expressing Nanog-EGFP at the condition of +2i (red, n = 20 cells), +LIF (blue, n = 20), −LIF (green, n = 23), −LIF+TSA (light green, n = 22), −LIF2d (purple, n = 21), and −LIF2d+TSA (pink, n = 19). Double asterisks and single asterisks indicate p-value < 0.05 and 0.05 ≤ p-value < 0.1, respectively, in Student's t-test.


Figure 3 ( C ) Typical example of dwell time histograms of Nanog - EGFP obtained within every 400 frames at frame periods of 50 ( green , n = 228 traces ) , 100 ( orange , n = 97 ) , 150 ( red , n = 102 ) , 250 ( magenta , n = 124 ) , and 450 ms ( blue , n = 157 ) in a nucleus in + LIF . ( D ) Dwell time histograms of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) obtained from all cell data ( n = 73 in + 2i , 65 in + LIF , and 66 cells in − LIF for Nanog - EGFP , and n = 57 in + 2i , 53 in + LIF , and 58 cells in − LIF for Oct4 - EGFP ) at frame periods of 50 , 100 , 150 , 250 , and 450 ms in a nucleus in + 2i ( red ) , + LIF ( blue ) , and − LIF conditions ( green ) . Values of koff were obtained by the global fitting approximation . ( E ) Histograms of dissociation rate of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) in an mESC in + 2i ( top ) , + LIF ( middle ) , and − LIF ( bottom ) conditions . Black line shows the fitting result with a normal distribution . ( F ) Dependence of expression level of Nanog - EGFP ( upper ) and Oct4 - EGFP ( lower ) on dissociation rate . Considering the distribution of the fluorescent intensity low ( blue ) , middle ( magenta ) , and high ( red ) were defined as cells showing the log value of the fluorescent intensity of < 1 . 9 , 2 . 0 - 2 . 3 , and > 2 . 4 [ a . u . ] , respectively , for Nanog - EGFP or < 1 . 9 , 2 . 0 - 2 . 2 , and < 2 . 3 [ a . u . ] , respectively , for Oct4 - EGFP . Bars and error bars are mean ± SD . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . For Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks indicate less than 0 . 05 of the p - value in Student ' s t - test .

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

Figure 3(C) Typical example of dwell time histograms of Nanog-EGFP obtained within every 400 frames at frame periods of 50 (green, n = 228 traces), 100 (orange, n = 97), 150 (red, n = 102), 250 (magenta, n = 124), and 450 ms (blue, n = 157) in a nucleus in +LIF.(D) Dwell time histograms of Nanog-EGFP (left) and Oct4-EGFP (right) obtained from all cell data (n = 73 in +2i, 65 in +LIF, and 66 cells in −LIF for Nanog-EGFP, and n = 57 in +2i, 53 in +LIF, and 58 cells in −LIF for Oct4-EGFP) at frame periods of 50, 100, 150, 250, and 450 ms in a nucleus in +2i (red), +LIF (blue), and −LIF conditions (green). Values of koff were obtained by the global fitting approximation.(E) Histograms of dissociation rate of Nanog-EGFP (left) and Oct4-EGFP (right) in an mESC in +2i (top), +LIF (middle), and −LIF (bottom) conditions. Black line shows the fitting result with a normal distribution.(F) Dependence of expression level of Nanog-EGFP (upper) and Oct4-EGFP (lower) on dissociation rate. Considering the distribution of the fluorescent intensity low (blue), middle (magenta), and high (red) were defined as cells showing the log value of the fluorescent intensity of < 1.9, 2.0-2.3, and > 2.4 [a.u.], respectively, for Nanog-EGFP or < 1.9, 2.0-2.2, and < 2.3 [a.u.], respectively, for Oct4-EGFP. Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks indicate less than 0.05 of the p-value in Student's t-test.


Figure 4 ( D , E ) Dependence of expression level of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) on Rc ( D ) or D ( E ) . Bars and error bars are mean ± SD . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . Of Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test .

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

Figure 4(D, E) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on Rc (D) or D (E). Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test.


Figure 5 ( C , D ) The average correlation index g ( r ) of Nanog - EGFP ( C ) and Oct4 - EGFP ( D ) in + 2i ( top ) , + LIF ( middle ) , and − LIF ( bottom ) . Insets are histograms of logarithm value of the intercept g0 ( magenta ) and the exponential decay constant ξ ( orange ) of single mESCs . Black lines in insets show the fitting results with a normal distribution . Error bars represent the standard deviation . N = 72 for + 2i , 56 for + LIF and 56 cells for − LIF of Nanog - EGFP , and N = 57 for + 2i , 53 for + LIF and 58 cells for − LIF of Oct4 - EGFP . ( E , F ) Dependence of expression level of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) on log10g0 ( E ) and log10ξ ( F ) . Error bars represent standard deviations . The definition of low ( blue ) , middle ( magenta ) , and high ( red ) in E and F were the same as that of Fig . 3F . Bars and error bars are mean ± SD . Of Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . Of Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test .

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

Figure 5(C, D) The average correlation index g(r) of Nanog-EGFP (C) and Oct4-EGFP (D) in +2i (top), +LIF (middle), and −LIF (bottom). Insets are histograms of logarithm value of the intercept g0 (magenta) and the exponential decay constant ξ (orange) of single mESCs. Black lines in insets show the fitting results with a normal distribution. Error bars represent the standard deviation. N = 72 for +2i, 56 for +LIF and 56 cells for −LIF of Nanog-EGFP, and N = 57 for +2i, 53 for +LIF and 58 cells for −LIF of Oct4-EGFP.(E, F) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on log10g0 (E) and log10ξ (F). Error bars represent standard deviations. The definition of low (blue), middle (magenta), and high (red) in E and F were the same as that of Fig. 3F. Bars and error bars are mean ± SD. Of Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.


Figure 6 ( B , A typical example of time developments of correlation index g ( r ) ( B ) in a mESC six hours after the removal of 2i and LIF . These were obtained from the same cell . C ) visualisation of the binding frequency of Nanog - EGFP per unit area ( density ) ( C ) in a mESC six hours after the removal of 2i and LIF . Colour in C corresponds to the density of Nanog dwell events ( events / μm2 ) within 2 , 000 frames . These were obtained from the same cell . Arrows indicate the emergence of hotspots of functioning Nanog , exhibiting obviously higher density than the others .

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

Figure 6(B, A typical example of time developments of correlation index g(r) (B) in a mESC six hours after the removal of 2i and LIF. These were obtained from the same cell.C) visualisation of the binding frequency of Nanog-EGFP per unit area (density) (C) in a mESC six hours after the removal of 2i and LIF. Colour in C corresponds to the density of Nanog dwell events (events/μm2) within 2,000 frames. These were obtained from the same cell. Arrows indicate the emergence of hotspots of functioning Nanog, exhibiting obviously higher density than the others.


Figure 7 ( C , D ) Plots of all data of cells categorised with expression level ( red , magenta , and blue ) and with the mean values ( green or cyan ) in each parameter in each condition of Nanog - EGFP ( C ) or Oct4 - EGFP ( D ) . The definition of low ( blue ) , middle ( magenta ) , and high ( red ) in + 2i , + LIF , and − LIF were the same as that of Fig . 3F , and those in − LIF2d were the same as B in this figure . Error bars represent standard deviations . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF , and 20 ( high ) , 20 ( middle ) and 19 cells ( low ) for − LIF2d . For Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF , and 16 ( high ) , 29 ( middle ) and 16 cells ( low ) for − LIF2d . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test . Note : the data obtained in − LIF2d could not simply compare with the others because the expression levels differed between them .

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

Figure 7(C, D) Plots of all data of cells categorised with expression level (red, magenta, and blue) and with the mean values (green or cyan) in each parameter in each condition of Nanog-EGFP (C) or Oct4-EGFP (D). The definition of low (blue), middle (magenta), and high (red) in +2i, +LIF, and −LIF were the same as that of Fig. 3F, and those in −LIF2d were the same as B in this figure. Error bars represent standard deviations. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, 20 (high), 26 (middle) and 20 cells (low) for −LIF, and 20 (high), 20 (middle) and 19 cells (low) for −LIF2d. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, 19 (high), 19 (middle) and 20 cells (low) for −LIF, and 16 (high), 29 (middle) and 16 cells (low) for −LIF2d. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test. Note: the data obtained in −LIF2d could not simply compare with the others because the expression levels differed between them.


Figure 8 ( A ) Correlation plots of log10g0 and koff of Nanog - EGFP ( left ) and Oct4 - EGFP ( centre ) and log10ξ and koff of Oct4 - EGFP ( right ) in 2i ( top ) , + LIF ( middle ) and − LIF ( bottom ) conditions . Each plot indicates the average value in a single cell . Lines are fitting results with a liner function . ( D ) Effect of 0 . 5 µM TSA addition on log10g0 of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) in + LIF ( filled ) and - LIF ( opened ) . Error bars represent standard deviations . For Nanog - EGFP , N = 65 for + LIF - TSA , 91 for + LIF + TSA , 66 for - LIF - TSA , and 88 cells for - LIF + TSA . For Oct4 - EGFP , N = 53 for + LIF - TSA , 42 for + LIF + TSA , 58 for - LIF - TSA , and 39 cells for - LIF + TSA . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test .

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

Figure 8(A) Correlation plots of log10g0 and koff of Nanog-EGFP (left) and Oct4-EGFP (centre) and log10ξ and koff of Oct4-EGFP (right) in 2i (top), +LIF (middle) and −LIF (bottom) conditions. Each plot indicates the average value in a single cell. Lines are fitting results with a liner function.(D) Effect of 0.5 µM TSA addition on log10g0 of Nanog-EGFP (left) and Oct4-EGFP (right) in +LIF (filled) and -LIF (opened). Error bars represent standard deviations. For Nanog-EGFP, N = 65 for +LIF -TSA, 91 for +LIF +TSA, 66 for -LIF -TSA, and 88 cells for -LIF +TSA. For Oct4-EGFP, N = 53 for +LIF -TSA, 42 for +LIF +TSA, 58 for -LIF -TSA, and 39 cells for -LIF +TSA. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.


Figure 2B ) Box - whisker plots of log2 - transformed peptide intensities across all 60 raw files . The color coding highlights the samples plate of origin . C ) Cumulative frequency curve [ % ] of the observed coefficient of variation ( CV ) of proteins that were identified and quantified with a minimum of three valid values within each day . Here , the ten raw files of each day are evaluated individually . The resulting median and average CV for each day are shown . D ) Pearson correlation heatmap of all 60 raw files and additional sixteen manually prepared HeLa SP3 samples . The displayed data are filtered for 75 % data completeness ( Table 1 ) . Please note the narrow scaling ( 1 - 0 . 94 ) .

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

Figure 2B) Box-whisker plots of log2-transformed peptide intensities across all 60 raw files. The color coding highlights the samples plate of origin.C) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified with a minimum of three valid values within each day. Here, the ten raw files of each day are evaluated individually. The resulting median and average CV for each day are shown.D) Pearson correlation heatmap of all 60 raw files and additional sixteen manually prepared HeLa SP3 samples. The displayed data are filtered for 75% data completeness (Table 1). Please note the narrow scaling (1-0.94).


Figure 4C ) Volcano plot showing differential expression analysis using Limma moderated t - statistics for the comparison of lepidic samples against all other samples . Proteins passing significance thresholds of - log10 p - value < 0 . 05 ( Benjamini - hochberg adjusted ) and an absolute log2 fold change of > 1 are highlighted in orange .

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Figure 4C) Volcano plot showing differential expression analysis using Limma moderated t-statistics for the comparison of lepidic samples against all other samples. Proteins passing significance thresholds of -log10 p-value < 0.05 (Benjamini-hochberg adjusted) and an absolute log2 fold change of >1 are highlighted in orange.


Figure 5B ) Correlation between mg wet tissue weight and the amount of protein extracted by ultrasonication . C ) Pearson correlation heatmap of peptide intensities across five replicates of each sample type ( heart , liver , kidney , HeLa cells ) with the corresponding average . D ) The relative number of identified and quantified proteins across the five replicates of each sample type . The average number of identified proteins and the standard deviation across five replicates is shown on top . E ) Cumulative frequency curve [ % ] of the observed coefficient of variation ( CV ) of proteins that were identified and quantified in at least three out of five replicates in each sample type . The resulting median and average CV for each sample type are shown .

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

Figure 5B) Correlation between mg wet tissue weight and the amount of protein extracted by ultrasonication.C) Pearson correlation heatmap of peptide intensities across five replicates of each sample type (heart, liver, kidney, HeLa cells) with the corresponding average.D) The relative number of identified and quantified proteins across the five replicates of each sample type. The average number of identified proteins and the standard deviation across five replicates is shown on top.E) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified in at least three out of five replicates in each sample type. The resulting median and average CV for each sample type are shown.


Figure 1A . Bclaf1 wild type ( WT ) and two Bclaf1 knockout HeLa cell lines ( KO - 1 and - 2 ) were pre - treated with DMSO or cycloheximide ( CHX , 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) together with CHX for 12 hours . Afterwards , the cells were subjected to Annexin V / 7AAD staining followed by flow cytometry analysis , all Annexin V positive cells were counted for analysis , or protein extractions followed by western blotting analysis . B , C . HeLa ( B ) or primary MEFs ( C ) were transfected with control ( siCtrl ) or two siRNAs against Bclaf1 ( siBclaf1 - 1 and - 2 ) and then treated and analyzed as described in A . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test . D , E . MEFs transfected with control ( siCtrl ) or siBclaf1 - 1 and / or siBclaf1 - 2 siRNAs were pre - treated with CHX ( 1 μg / ml ) / z - VAD ( 20 μM ) ( D ) or SM - 164 ( 100 nM ) / z - VAD ( 20 μM ) ( E ) for 30 min , and then added TNF ( 10 ng / ml ) for indicated times . The cells were subjected to Annexin V / 7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells ( up ) , or protein extractions and western blotting analysis at indicated time periods ( down ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

Figure 1A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


Figure 2A , B . Cell lysates of HeLa Bclaf1 WT and KO cells ( A ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( B ) treated with TNF ( 10 ng / ml ) for indicated time periods were analyzed by western blotting with the indicated antibodies . C , D . HeLa Bclaf1 WT and KO cells ( C ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( D ) were treated with TNF ( 10 ng / ml ) for the indicated time periods , and then subject to nucleus - cytoplasm fractionation as described in materials and methods followed by western blotting . Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions , respectively .

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

Figure 2A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.


Figure 3B . MEFs treated with TNF ( 10 ng / ml ) for 4 hours were subject to FADD immunoprecipitation , and then analyzed by western blotting . G , H . HeLa cells transfected with siRNAs against Bclaf1 or c - FLIP were pretreated with CHX ( 1 μg / ml ) for 30 min and then stimulated with TNF ( 10 ng / ml ) in the presence of CHX for 12 hours followed by Annexin V / 7AAD staining and flow cytometry analysis ( G ) , or protein extractions followed by western blotting analysis ( H ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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Figure 3B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.


Figure 4A - F . Bclaf1 WT , KO - 1 and - 2 HeLa cells ( A and D ) , as well as HeLa ( B and E ) and primary MEFs ( C and F ) which were transfected with siCtrl or siBclaf1 - 1 and - 2 were treated with TNF ( 10 ng / ml ) for 6 hours followed by western blotting analysis ( A - C ) , or for 4 hours followed by total RNA extraction and RT - PCR analysis ( D - F ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . H , I . CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa - Flag - Bclaf1 cells treated without ( H ) or with TNF ( I ) . The regions amplified by RT - PCR were delineated . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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Figure 4A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


Figure 5B . CFLAR promoter were co - transfected with Bclaf1 or / and p50 into HeLa - Bclaf1 - KO cells , and the luciferase activity was measured as described before . E , HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre - treated with DMSO or CHX ( 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) for 12 hours . Cells were lysed and then analyzed by western blottingG . CHIP analysis of the CFLAR promoter in HeLa - Flag - Bclaf1 cells . Immunoprecipitation was performed using anti - Flag and control IgG antibodies followed by RT - PCR analysis . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way or Two - way ANOVA test .

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Figure 5B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blottingG. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.


Figure 6A . HeLa - Flag - Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF ( 10 ng / ml ) for 2 hours were separated into the cytoplasmic and the nuclear fraction . Endogenous Flag - Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting . B . HeLa - Flag - Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF ( 10 ng / ml ) for 2 hours . The nuclear fraction was isolated and subjected to immunoprecipitation with an anti - Flag or control IgG antibody , followed by western blotting analysis . D . CFLAR FL promoter were co - transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa - Bclaf1 - KO cells . The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant . One - way ANOVA test .

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Figure 6A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.


Figure 7siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for Bclaf1 ( B ) and Hematoxylin - and - eosin staining ( C ) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis ( B ) . Hematoxylin - and - eosin staining showing the morphological change ( C ) . Villus height and crypt depth were measured ( D ) . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μm in B , 100 μm in C . siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 ( E ) , phospholated RIPK3 ( F ) CC3 + and p - RIPK3 + positive cells in five fields per intestine were quantified . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μmsiRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for western blotting analysis ( G ) . HIEC - 6 cells were transfected siCtrl or siBclaf1 - 1 and - 2 . Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis ( I and J )

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Figure 7siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μmsiRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G).HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J)


Figure 1 A Electron micrograph of unstained UMOD filament sheets in human urine . The inset highlights a double helix - like structure resulting from juxtaposition of two individual filaments . Scale bars : 50 nm and 10 nm ( inset ) . B , C " Tree " front view and " zig - zag " side view of purified native UMOD filaments , imaged using a Volta phase plate . Scale bars : 10 nm . D , E Front and side views of UMODe filaments , showing absence of branches . Scale bars : 10 nm .

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Figure 1 A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset). B, C "Tree" front view and "zig-zag" side view of purified native UMOD filaments, imaged using a Volta phase plate. Scale bars: 10 nm. D, E Front and side views of UMODe filaments, showing absence of branches. Scale bars: 10 nm.


Figure 4 A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that , unlike wt full - length UMOD , mutants ZP - N ( R415A ) or ZP - C ( ΔFA ) do not assemble into filaments . Scale bar : 50 µm . B - G Immunofluorescence of unpermeabilized cells co - expressing FLAG - tagged wt UMOD ( green ) and the indicated HA - tagged isoforms ( red ) . UMOD R415A does not incorporate into polymers that only contain wt protein ( C ) . UMOD ΔFA has a dominant negative effect on wt protein polymerization ( D ) that is rescued in a double mutant carrying both ZP - N and ZP - C mutations ( F ) . Similarly , the dominant negative effect of a CCS mutation ( 4A ) that prevents EHP dissociation ( E ) is suppressed by the introducing the R415A mutation in the 4A isoform ( G ) . Scale bar : 50 µm . H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP ( UMOD - CCS ) shows that wt forms intracellular polymers whereas polymerization interface mutants do not . The intracellular polymers are localized in the endoplasmic reticulum ( ER ) , as shown by co - staining with the KDEL sequence used as ER marker . Scale bar : 10 µm .

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Figure 4 A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that, unlike wt full-length UMOD, mutants ZP-N (R415A) or ZP-C (ΔFA) do not assemble into filaments. Scale bar: 50 µm.  B-G Immunofluorescence of unpermeabilized cells co-expressing FLAG-tagged wt UMOD (green) and the indicated HA-tagged isoforms (red). UMOD R415A does not incorporate into polymers that only contain wt protein (C). UMOD ΔFA has a dominant negative effect on wt protein polymerization (D) that is rescued in a double mutant carrying both ZP-N and ZP-C mutations (F). Similarly, the dominant negative effect of a CCS mutation (4A) that prevents EHP dissociation (E) is suppressed by the introducing the R415A mutation in the 4A isoform (G). Scale bar: 50 µm.  H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP (UMOD-CCS) shows that wt forms intracellular polymers whereas polymerization interface mutants do not. The intracellular polymers are localized in the endoplasmic reticulum (ER), as shown by co-staining with the KDEL sequence used as ER marker. Scale bar: 10 µm.


Figure 5 A Analytical SEC of UFE HCE / LCE - digests produces two protein peaks , F1 and F2 . Reducing SDS - PAGE of SEC purified UFE digests with indicated molecular weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE . native - PAGE of SEC purified UFE digests with indicated molecular weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE . D , E TOF - MS spectrograms of purified F2 ( D ) and F1 ( E ) products . Round - shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI - 3 ( green ) or the separated ZP - N and ZP - C domains of LCE - cleaved ZI - 1 , 2 ( purple ) . F , G TOF - MS spectrograms of cross - linked products of F2 ( F ) and F1 ( G ) , with domain symbols indicating the deduced subunit composition of cross - linked products . I Electron micrograph of negatively - stained fish egg coat filaments . On the right are representative 2D classes , indicating a separation of ~ 65 Å between repeating units within the polymers ( white arrows ) . Scale bars : 100 nm ( left panel ) and 10 nm ( bottom right panel ) .

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

Figure 5 A Analytical SEC of UFE HCE/LCE-digests produces two protein peaks, F1 and F2. Reducing SDS-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE. native-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE. D, E TOF-MS spectrograms of purified F2 (D) and F1 (E) products. Round-shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI-3 (green) or the separated ZP-N and ZP-C domains of LCE-cleaved ZI-1,2 (purple).F, G TOF-MS spectrograms of cross-linked products of F2 (F) and F1 (G), with domain symbols indicating the deduced subunit composition of cross-linked products. I Electron micrograph of negatively-stained fish egg coat filaments. On the right are representative 2D classes, indicating a separation of ~65 Å between repeating units within the polymers (white arrows). Scale bars: 100 nm (left panel) and 10 nm (bottom right panel).


Figure 6 A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl ( top ) . Analysis of this material by correlation averaging reveals a periodic pattern ( bottom ) with a two - fold symmetry axis perpendicular to the plane of the sheet ( white symbol ) . Scale bars : 250 Å ( top ) and 125 Å ( bottom ) .

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Figure 6 A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl (top). Analysis of this material by correlation averaging reveals a periodic pattern (bottom) with a two-fold symmetry axis perpendicular to the plane of the sheet (white symbol). Scale bars: 250 Å (top) and 125 Å (bottom).


Figure 1B . Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation ( IP ) and associated proteins were detected by immunoblotting ( IB ) . Mock includes beads only control . C . Flag - tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells . Flag - WDTC1 complexes were immunoprecipitated with anti - FLAG and their associated proteins were detected by immunoblotting as indicated ; EV , empty vector control . E . 293T cells were transfected with HA - ubiquitin along with various combinations of plasmid and siRNA as indicated . Transfected cells were lysed under denaturing conditions to obtain whole cell extracts ( WCE ) and immunoprecipitated with anti - FLAG . WDTC1 ubiquitylation was evaluated by immunoblotting as indicated . F . 293T cells transiently expressing Flag - WDTC1 were transfected with scramble ( scrm ) siRNA or siRNAs against DDB1 , CUL4A and CUL4B . Flag - WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated .

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Figure 1B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control.C. Flag-tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control.E. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated.F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.


Figure 2A . 3T3 - L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days . Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti - WDTC1 followed by immunoblot analyses with indicated antibodies . Fatty acid synthase ( FAS ) levels were monitored to evaluate induction . B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Adipogenesis was assessed by Oil Red O staining ( ORO ) ( B ) ; plate ( top ) and microscopic ( bottom ) views , 0 . 1 mm scale bar . B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Triglyceride levels were quantified by an enzymatic assay ( C ) . B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Adipogenic gene expression was evaluated by RT - qPCR analysis ( D ) .

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Figure 2A. 3T3-L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction.B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar.B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C).B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenic gene expression was evaluated by RT-qPCR analysis (D).


Figure 3A . Schematic of WDTC1 knockdown - rescue lentiviral vector ( top ) . 3T3 - L1preadipocytes were lentivirally transduced to generate stable cells expressing a non - specific ( NS ) shRNA control , shWDTC1 targeting endogenous Wdtc1mRNA , or cells simultaneously expressing shWDTC1 and shRNA - resistant WDTC1 ; bottom , confirmation of knockdown and WDTC1 ectopic expression . B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by ORO ( B ) B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by triglyceride quantitation ( C ) B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by adipogenic gene expression ( D ) .

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Figure 3A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression.B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B)B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C)B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by adipogenic gene expression (D).


Figure 4A . Induced 3T3 - L1 cells stably expressing Flag - WDTC1 were subjected to subcellular fractionation . Aliquots of total cell extract ( TCE ) and subcellular fractions were analyzed by immunoblot as indicated ; fraction purity was assessed by α - Tubulin and histone H3 antibodies . B . Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag - WDTC1 and HA tagged core histones were immunoprecipitated ( IP ) with anti - FLAG and immunoblotted ( IB ) as indicated . The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk . C . Soluble chromatin extracts were prepared from preadipocyte or induced 3T3 - L1 cells stably expressing control ( EV ) and Flag - WDTC1 proteins . H2A monoubiquitylation was detected by H2AK119ub1 - specific antibody and total H2A served as loading control . Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control . D . Affinity purified Flag - WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays . Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining ( left panel ) . In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins . Reaction products were resolved by SDS - PAGE , and ubiquitylatedH2A was detected by anti - H2AK119ub1 ( right panel ) . E . In vitro ubiquitylation assays were performed with purified wild - type and mutant histone proteins as substrates . H2A ubiquitylation was assessed by immunoblotting with anti - ubiquitin and various antibodies as indicated .

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Figure 4A. Induced 3T3-L1 cells stably expressing Flag-WDTC1 were subjected to subcellular fractionation. Aliquots of total cell extract (TCE) and subcellular fractions were analyzed by immunoblot as indicated; fraction purity was assessed by α-Tubulin and histone H3 antibodies.B. Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag-WDTC1 and HA tagged core histones were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) as indicated. The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk.C. Soluble chromatin extracts were prepared from preadipocyte or induced 3T3-L1 cells stably expressing control (EV) and Flag-WDTC1 proteins. H2A monoubiquitylation was detected by H2AK119ub1-specific antibody and total H2A served as loading control. Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control.D. Affinity purified Flag-WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays. Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining (left panel). In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins. Reaction products were resolved by SDS-PAGE, and ubiquitylatedH2A was detected by anti-H2AK119ub1 (right panel).E. In vitro ubiquitylation assays were performed with purified wild-type and mutant histone proteins as substrates. H2A ubiquitylation was assessed by immunoblotting with anti-ubiquitin and various antibodies as indicated.


Figure 1A . Four representative images of RHOB immunostaining in human non - small - cell lung cancers . Figures correspond to the percentage of tumor analyzed . B . Characteristics of 96 patients with an EGFR - activating mutation who were treated with EGFR - TKI . Each column corresponds to one patient . Data include general characteristics ( stage at diagnosis , age , sex , tobacco usage , position of EGFR mutation , TKI type ) , progression - free survival ( PFS ; lower graph ) and intensity of RHOB staining as determined by immunohistochemistry ( null : 0 , weak : + , moderate : + + and high : + + + ) . C . Representative scans of patients with low or high RHOB - expressing lung tumors , before and after erlotinib treatment . Red arrows indicate lung tumors . D . Progression - free survival of erlotinib - treated patients with EGFR - mutated lung tumors , according to RHOB expression , assessed by immunohistochemistry ( low RHOB group = negative + weak staining ; high RHOB group = moderate + high staining ) . E . Progression - free survival of patients who received EGFR - TKi as first - line therapy ( n = 63 ) . F . Progression - free survival of patients who received EGFR - TKi as 2nd - line ( n = 28 ) , 3rd - line ( n = 3 ) or 4th - line ( n = 2 ) therapy . G . RHOB immunostaining score evolution in EGFR - mutated lung tumors before treatment and after EGFR - TKI relapse .

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

Figure 1A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed.B. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++).C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).E. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy.G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.