Figure 1A ) We identified previously unknown telomere regulators in fission yeast using the haploid S . pombe whole - genome gene deletion library including ssu72 + ( SPAC3G9 . 04 ) . B ) Telomere length of wt , ssu72Δ and ssu72 - C13S ( point mutation at the phosphatase active site ) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe . C ) Ssu72 recruitment to telomeres is cell cycle regulated . Ssu72 was myc - tagged at the 3 ' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains . Septa formation was used proxy for S - phase . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to ssu72 + control samples . Error bars represent standard error of the mean ( SEM ) . D ) Telomere length of ssu72Δ is dependent on telomerase . Diploid strains with the appropriate genotypes were sporulated and trt1 ∆ ssu72 ∆ double mutants were streaked for multiple passages ( triangle indicates increased number of generations ) . E ) Telomerase is recruited to telomeres in the absence of Ssu72 . ChIP analysis for Trt1 - myc in wt and ssu72 ∆ was performed as described in material and methods using a non - tagged strain as a control . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) . F ) Telomerase activator Ccq1 is hyperphosphorylated in ssu72 ∆ cells . rap1 ∆ cells were used as positive control for Ccq1 phosphorylation status . Western blots were performed using Ccq1 - flag tagged strains .

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 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 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 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

Figure 1A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe. C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations).E) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.


Figure 2A ) ssu72Δ telomeres present longer G - rich overhangs than wt and rif1 ∆ . In - gel hybridization in native and denaturing conditions was labelled with a radiolabelled C - rich telomere probe and quantified for ssDNA at the telomeres . n = 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) . B ) RPA ( Rad11 - GFP ) is enriched at ssu72 ∆ telomeres . Colocalization of Rad11 - GFP with Taz1 - mCherry , used as a telomere marker was performed in wt and ssu72 ∆ cells ; n = 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) . More than 1000 cells were analyzed for each genotypeC ) ssu72 + controls telomere length independently of rif1 + . Epistasis analysis of telomere length of ssu72 ∆ and ssu72 - C13S ( catalytically inactive mutant ) with rif1 ∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe . D ) ssu72 + and stn1 + regulate telomere length in the same genetic pathway . Epistasis analysis of ssu72 ∆ and stn1 - 75 performed by Southern blotting of ApaI digested DNA using a telomeric probe . Two independently generated ssu72 ∆ stn1 - 75 double mutants are shown .

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 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 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

Figure 2A) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotypeC) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.


Figure 3A ) Ssu72 is required for telomere recruitment of Stn1 in late S phase . ChIP analysis of stn1 - myc in wt and ssu72 ∆ cells was performed in synchronized cdc25ts cells . n ≥ 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to ssu72 + control samples . Error bars represent standard error of the mean ( SEM ) . B ) 2D - gel analysis of NsiI telomeric fragments of wt and ssu72 ∆ strains . Smart ladder from Eurogentec was used for DNA size measurement . C ) Serine 74 substitution to a phosphomimetic aspartate amino acid ( stn1 - S74D ) is sufficient to confer ssu72 ∆ telomere defects . Telomere length epistasis analysis of ssu72 ∆ and stn1 - S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe . E ) Similar to ssu72 ∆ mutants , stn1 - S74D is defective in telomere recruitment . ChIP analysis of stn1 - myc and stn1 - S74D - myc using a non - tagged strain as a control . n = 3 ; * p ≤ 0 . 05 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) .

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 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 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

Figure 3A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.E) Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


Figure 4A ) ssu72 + and pol1 + regulate telomere length in the same genetic pathway . Epistasis analysis of ssu72 ∆ and pol1 - 13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe . B ) Stn1 - Pol1 interaction requires Ssu72 phosphatase . Immunoprecipitation experiments of Pol1 - Flag with Stn1 - Myc was preformed both in wt and ssu72 ∆ mutants . C ) Immunoprecipitation of Ten1 - Flag with Stn1 - Myc in either wt or ssu72 ∆ was performed as control for Stn1 - Ten1 complex integrity . D ) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants . Multi - copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells .

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 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

Figure 4A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe.B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants. C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity. D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.


Figure 5A ) Telomere elongation of SSU72 down - regulated cells is telomerase dependent . Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 ( CDS and UTR regions ) and Luciferase ( Luc ) as control for shRNA . Knockdown efficiencies were determined by RT - qPCR using specific primers against hSSU72 . B ) Telomere elongation of SSU72 down - regulated cells is epistatic with STN1 downregulation . TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA , SSU72 shRNA ( against UTR ) , STN1 shRNA or double SSU72 shRNA / STN1 shRNA . Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis . C ) hSSU72 down - regulation results in multi - telomeric signals ( MTS ) that are dependent on DNA replication . Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid . Telomere FISH was carried out using a PNA - telomere probe . MTS are represented by arrows . Quantification of MTS per metaphase : n = 4 ; * * p ≤ 0 . 01 * * * * p ≤ 0 . 0001 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) .

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 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

Figure 5A) Telomere elongation of SSU72 down-regulated cells is telomerase dependent. Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 (CDS and UTR regions) and Luciferase (Luc) as control for shRNA. Knockdown efficiencies were determined by RT-qPCR using specific primers against hSSU72.B) Telomere elongation of SSU72 down-regulated cells is epistatic with STN1 downregulation. TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA, SSU72 shRNA (against UTR), STN1 shRNA or double SSU72 shRNA/STN1 shRNA. Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis.C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


Figure 6A ) hSSU72 downregulation results in telomere DNA damage foci ( TIF ) . Cells with indicated shRNAs were fixed and IF - FISH analysis was performed using a 53BP1 antibody and a PNA - telomere probe . ( Right ) Quantification of cells with more than 5 telomeric 53BP1 foci : n = 3 ; * * p ≤ 0 . 01 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) . B ) hSSU72 is required for efficient loading of hSTN1 at human telomeres . ChIP was preformed using anti - FLAG antibodies and Southern blotting was performed using a human telomeric probe . Quantification of 3 independent ChIP experiments * p ≤ 0 . 05 * * p ≤ 0 . 01 based on a two - tailed Student ' s t - test to control sample . Error bars represent standard error of the mean ( SEM ) .

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 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

Figure 6A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).B) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


Figure 1A Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles ( HF ) for Integrin α6 ( Itgα6 , red ) , active Caspase 3 ( aCas3 , green ) and DAPI ( blue ) in control ( Ctr ) and Bcl - 2 inhibitor ( ABT - 199 ) treated mice . B : bulge , CH : club hair , DP : dermal papilla , HF : hair follicle , HG : secondary hair germ . Scale bar , 50 µm B Quantification of backskin HF with aCas3 + cells in bulge , hair germ and dermal papilla of Ctr and ABT - 199 treated mice ( n = 4 mice biological replicates ; mean ± standard deviation ( SD ) ; * * * p < 0 . 001 , ns = not significant , two - way ANOVA test ) . nd = not detected . C , D Treatment scheme , FACS plot ( C ) and quantification ( D ) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution ( Ctr ) or ABT - 199 ( n = 6 biological replicates ; mean ± standard deviation ( SD ) ; * * p < 0 . 01 , ns = not significant , two - tailed unpaired Student ' s t - test ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs and n - B ( non - bulge epidermal cells ) . * * p < 0 . 005 .

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 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

Figure 1A Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles (HF) for Integrin α6 (Itgα6, red), active Caspase 3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG: secondary hair germ. Scale bar, 50 µm B Quantification of backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected. C,D Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.


Figure 2B Treatment scheme of experimental timeline and histology of backskin sections of control ( Ctr ) and ABT - 199 treated mice . Scale bar , 200 µm . D qRT - PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT - 199 treated and control ( Ctr ) mice at P77 ( n = 3 biological replicates ; mean ± standard error of mean ( SEM ) ; * * p < 0 . 01 , ns = not significant , two - tailed unpaired Student ' s t - 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 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

Figure 2B Treatment scheme of experimental timeline and histology of backskin sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.D qRT-PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).


Figure 3C qRT - PCR for mRNA expression of the anoikis - related markers Bim and Bmf in sorted n - B ( non - bulge ) , bBSC ( basal BSCs ) and sbBSC ( suprabasal BSCs ) ( n = 3 biological replicates ; mean ± standard error of mean ( SEM ) ; * p < 0 . 05 , ns = not significant , two - 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

Figure 3C qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).


Figure 4C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control ( Ctr ) and Bcl - 2EOE mice ( n = 3 biological replicates ; mean ± standard deviation ( SD ) ; ns = not significant , two - tailed unpaired Student ' s t - test ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs . D Immunofluorescence staining of telogen backskin HF for BrdU incorporation ( 1 h pulse , green ) and DAPI ( blue ) in 8 week old control ( Ctr ) and Bcl - 2EOE mice . Scale bar , 50 µm .

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 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

Figure 4C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control (Ctr) and Bcl-2EOE mice (n=3 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs.D Immunofluorescence staining of telogen backskin HF for BrdU incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.


Figure 5A Histology of P19 catagen backskin HF of control ( Ctr ) and Bcl - 2EOE mice . RS : retracting strand , Scale bar , 50 µm . B Analysis of length and width of retracting strands of control ( Ctr ) and Bcl - 2EOE mice ( n = 4 ; multiple measurements / biological replicate ; whiskers : min and max ; boxes : median ± first and third quartile ; * * * * p < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control ( Ctr ) and Bcl - 2EOE mice ( n = 4 biological replicates ) . Dashed lines : retracting strands . Scale bar , 100 µm . D Quantification of aCas3 + cells in the RS of P19 Ctr and Bcl - 2EOE mice ( n = 4 biological replicates ; mean ± standard deviation ( SD ) ; * * * * p < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . HF : hair follicle . F , G qRT - PCR for mRNA expression of the quiescence - related markers Bmp6 ( F ) and Fgf18 ( G ) in epidermis of control ( Ctr ) and Bcl - 2EOE mice ( P27 ) , ( n = 4 biological replicates ; mean ± standard error of mean ( SEM ) ; * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired Student ' s t - 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 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 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 5A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm. B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test). C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm. D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle. F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).


Figure 6B Immunofluorescence staining of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , activated Caspase 3 ( aCas3 , red ) and DAPI ( blue ) . Note increased number of aCas + cells in the bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous gland . Scale bar , 100µm . D Immunofluorescence staining of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , BrdU ( 1 h pulse , red ) and DAPI ( blue ) . Note increased number of BrdU + cells in the bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous gland . Scale bar ,

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 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

Figure 6B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm


Figure 7B , C Tumour incidence ( B ) and frequency ( C ) in Ctr , Bcl - 2EOE , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice ( n = 10 biological replicates ; mean ± standard error of mean ( SEM ) ) . D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice . Scale bar , 100µm .

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

Figure 7B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)).D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.


Figure 1 ( F ) Representative images of germ cells immunostained for SYCP3 ( green ) , DMC1 ( red ) , and DDX4 ( magenta ) at each meiotic sub - stage . Nuclear DAPI staining is shown in white . The sub - stages of meiosis were defined based on the staining patterns of SYCP3 , DMC1 , and DAPI as described in Materials and Methods . ( G ) Percentages of germ cells at each meiotic sub - stage as defined in Fig 1F . The average percentages in the outer or inner ovarian cortex on each meiotic sub - stage were calculated from at least three fetal ovaries [ 8 wpf ( n = 4 ) , 10 / 12 / 14 / 16 / 18 wpf ( n = 3 ) ] .

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

Figure 1(F) Representative images of germ cells immunostained for SYCP3 (green), DMC1 (red), and DDX4 (magenta) at each meiotic sub-stage. Nuclear DAPI staining is shown in white. The sub-stages of meiosis were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. (G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F. The average percentages in the outer or inner ovarian cortex on each meiotic sub-stage were calculated from at least three fetal ovaries [8 wpf (n=4), 10/12/14/16/18 wpf (n=3)].


Figure 2 ( C ) IF for oogonia ( POU5F1 , TFAP2C ) , meiotic ( SYCP3 , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers in the transplanted cy rOvaries . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper - right magnified images of each panel show the expression of each marker ( green ) co - stained with DDX4 ( magenta ) . The lower - right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . The numbers written in the upper - left corner indicate weeks after transplantation ( w - ptp ) . Scale bar = 20 μm . ( D ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of cy rOvaries at 3 − 15 w - ptp [ 3 w - ptp ( n = 3 ) , 6 / 9 / 12 w - ptp ( n = 4 ) , 15 w - ptp ( n = 6 ) ] . The average values with SDs are shown . ( E ) H & E staining of transplanted cy rOvaries at 21 w - ptp . Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN / Slc mice . K , mouse kidney . Scale bars = 200 μm ( left ) and 100 μm ( right ) .

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

Figure 2(C) IF for oogonia (POU5F1, TFAP2C), meiotic (SYCP3, SYCP1), and oogenic (ZP3, TP63) markers in the transplanted cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of each marker (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate weeks after transplantation (w-ptp). Scale bar = 20 μm. (D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3−15 w-ptp [3 w-ptp (n=3), 6/9/12 w-ptp (n=4), 15 w-ptp (n=6)]. The average values with SDs are shown. (E) H&E staining of transplanted cy rOvaries at 21 w-ptp. Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN/Slc mice. K, mouse kidney. Scale bars = 200 μm (left) and 100 μm (right).


Figure 3 ( E ) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper - right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower - right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . The numbers written in the upper - left corner indicate the period of IVC ( w - ivc ) . Scale bar = 20 μm . ( F ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of cy rOvaries at 3 - 15 w - ivc [ 3 / 6 / 9 / 12 w - ivc ( n = 3 ) , 15 w - ivc ( n = 3 or 4 ) ] .

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 3(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm. (F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)].


Figure 5 ( A ) Gross appearance ( a ) , H & E staining ( b ) , and IF ( c ) for FOXL2 / DDX4 / DAPI of the human in vivo female gonads ( 11W , left column ) and the cultured rOvaries at 7 and 14 w - ivc . FOXL2 , granulosa cell marker ; DDX4 , germ cell marker ; DAPI , nucleus . Scale bars = 1 mm ( a , left ) , 200 μm ( a , middle / right ) , 50 μm ( b ) , and 20 μm ( c ) . g . a . , gestational age . IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers for human in vivo female gonad ( 11W , B ) Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers human in vitro cultured rOvaries at 14 w - ivc ( C ) . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . ( D ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of human in vivo female gonads ( 11W ) and human cultured rOvaries at 7 and 14 w - ivc . The mean values from two biological replicates are shown . Analyses for human germ cells by SC3 - seq . ( E ) UHC for human germ cells in vivo and in rOvaries with all expressed genes ( 20 , 048 genes ) and a heatmap of the expression levels of selected marker genes . Color bars under the dendrogram show the in vivo stage ( 1st bar ) , the period of IVC ( 2nd bar ) , and the cell type ( 3rd bar ) . The heatmap color - coding is as indicated . The color coding in bars is as indicated in Fig 5F .

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 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 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 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 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

Figure 5(A) Gross appearance (a), H&E staining (b), and IF (c) for FOXL2/DDX4/DAPI of the human in vivo female gonads (11W, left column) and the cultured rOvaries at 7 and 14 w-ivc. FOXL2, granulosa cell marker; DDX4, germ cell marker; DAPI, nucleus. Scale bars = 1 mm (a, left), 200 μm (a, middle/right), 50 μm (b), and 20 μm (c). g.a., gestational age.IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers for human in vivo female gonad (11W, B) Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers human in vitro cultured rOvaries at 14 w-ivc (C). Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).(D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of human in vivo female gonads (11W) and human cultured rOvaries at 7 and 14 w-ivc. The mean values from two biological replicates are shown.Analyses for human germ cells by SC3-seq. (E) UHC for human germ cells in vivo and in rOvaries with all expressed genes (20,048 genes) and a heatmap of the expression levels of selected marker genes. Color bars under the dendrogram show the in vivo stage (1st bar), the period of IVC (2nd bar), and the cell type (3rd bar). The heatmap color-coding is as indicated. The color coding in bars is as indicated in Fig 5F.


Figure 6Analyses performed by 10X scRNA - seq . ( A ) UMAP plot of germ cells from six 10X scRNA - seq datasets ( two for humans [ this study and ( Chitiashvili et al . , 2020 ) ] , one for monkeys [ this study ] , and three for mice [ ( Ge et al . , 2021 , Niu & Spradling , 2020 , Zhao et al . , 2020 ) ] ) integrated by CCA . Meiotic sub - stages were defined after CCA followed by Louvain clustering . Colors indicate the computationally assigned meiotic sub - stages defined by the expression levels of key meiotic markers conserved in all three species ( see Materials and Methods ) . The color - coding is as indicated . M , mitotic ; PL , pre - leptotene ; L , leptotene ; Z , zygotene ; P , pachytene ; D , diplotene . ( B ) The UMAP plot highlighting cells from each dataset . The color - coding for each developmental stage is as indicated . ( I ) IF analyses of CLGN with TP63 ( oogenic marker ) and DDX4 ( germ cell marker ) in a 13 - year - old human oocyte of a primordial follicle . Nuclear DAPI staining is shown in white .

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 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

Figure 6Analyses performed by 10X scRNA-seq. (A) UMAP plot of germ cells from six 10X scRNA-seq datasets (two for humans [this study and (Chitiashvili et al., 2020)], one for monkeys [this study], and three for mice [(Ge et al., 2021, Niu & Spradling, 2020, Zhao et al., 2020)]) integrated by CCA. Meiotic sub-stages were defined after CCA followed by Louvain clustering. Colors indicate the computationally assigned meiotic sub-stages defined by the expression levels of key meiotic markers conserved in all three species (see Materials and Methods). The color-coding is as indicated. M, mitotic; PL, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene. (B) The UMAP plot highlighting cells from each dataset. The color-coding for each developmental stage is as indicated.(I) IF analyses of CLGN with TP63 (oogenic marker) and DDX4 (germ cell marker) in a 13-year-old human oocyte of a primordial follicle. Nuclear DAPI staining is shown in white.


Figure 7 ( C ) The Chr . 10 : A and X : A ratios ( top ) , and XIST expression ( bottom ) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3 - seq .

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

Figure 7(C) The Chr.10:A and X:A ratios (top), and XIST expression (bottom) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3-seq.


Figure 1Time - dependent acceptor fluorescence changes due to FRET during co - translational insertion of acceptor - labeled LepB75 into donor - labeled translocon .

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

Figure 1Time-dependent acceptor fluorescence changes due to FRET during co-translational insertion of acceptor-labeled LepB75 into donor-labeled translocon.


Figure 2Analysis of LepB94 translation products on SDS - PAGE . Translation products are visualized by the fluorescence of N - terminal Atto655 . Pausing intermediates are indicated P1 and P2 . Fitting of time courses ( filled circles ) of the translation of LepB mRNA constructs of increasing length ( Appendix Fig S3 ) by delay - exponential functions ( red lines ) . Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by the fluorescence of the Atto655 acceptor placed at the N - terminus of the nascent peptide . Fits ( red lines ) were obtained by global fitting Intrinsic fluorescence intensities ( IFI ) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting ( red squares ) . Confidence contours ( shaded areas ) were calculated using the Fitspace

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

Figure 2Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2. Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines).  Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm


Figure 3 Time course of EmrD135 translation . Translation was monitored by SDS - PAGE as in Fig 2A . Time course of EmrD ( - ) 135 translation . Stopped - flow time courses for translocon insertion of TM1 of EmrD ( black ) and EmrD ( - ) ( blue ) with mRNA constructs of varying chain length ( 50 to 135 aa ) . The FRET acceptor ( Atto655 ) was placed at the N - terminus of the nascent peptide , the donor ( Atto488 ) at position 111 ( donor - in ) of SecY . The results of global fitting are indicated ( red lines ) . Intrinsic fluorescence intensities ( IFIs ) calculated for EmrD ( black ) and EmrD ( - ) ( blue ) . Error margins ( shaded areas ) are calculated using the Fitspace algorithmStopped - flow traces for co - translational insertion of LepB94 ( red , from Fig 2 ) , EmrD135 ( black , from Fig 4E ) , and EmrD ( - ) 135 ( blue , from Fig 4E ) , rescaled according to the translation efficiency .

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

Figure 3 Time course of EmrD135 translation. Translation was monitored by SDS-PAGE as in Fig 2A. Time course of EmrD(-)135 translation.  Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines). Intrinsic fluorescence intensities (IFIs) calculated for EmrD (black) and EmrD(-) (blue). Error margins (shaded areas) are calculated using the Fitspace algorithmStopped-flow traces for co-translational insertion of LepB94 (red, from Fig 2), EmrD135 (black, from Fig 4E), and EmrD(-)135 (blue, from Fig 4E), rescaled according to the translation efficiency.


Figure 4 A , B , C LepB ( A ) , EmrD ( B ) , and EmrD ( - ) ( C ) are translated for the times indicated and subjected to PK treatment ( Materials and Methods ) . Sensitivity to PK in the absence of SecYEG ( black circles ) or in the presence of SecYEG ( red squares ) , and co - translational translocon insertion monitored by FRET ( blue trace ) are depicted . Error bars represent standard deviations ( n ≥ 3 ) and the corresponding lines represent fits to delay - exponential functions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B, C LepB (A), EmrD (B), and EmrD(-) (C) are translated for the times indicated and subjected to PK treatment (Materials and Methods). Sensitivity to PK in the absence of SecYEG (black circles) or in the presence of SecYEG (red squares), and co-translational translocon insertion monitored by FRET (blue trace) are depicted. Error bars represent standard deviations (n≥3) and the corresponding lines represent fits to delay-exponential functions.


Figure 1 ( C ) Western blot analysis of STAT2 protein levels in mock - , WT - RCMV - E - , ΔE27 - RCMV - E - or UV - inactivated RCMV - E - infected REF . 24 h p . i . , cells were either left untreated or treated for 5 . 5 h with DMSO , 2 . 5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition . IE1 and β - tubulin served as infection and loading controls , respectively .

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

Figure 1(C) Western blot analysis of STAT2 protein levels in mock-, WT-RCMV-E-, ΔE27-RCMV-E- or UV-inactivated RCMV-E-infected REF. 24 h p.i., cells were either left untreated or treated for 5.5 h with DMSO, 2.5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition. IE1 and β-tubulin served as infection and loading controls, respectively.


Figure 2 ( A , B ) GF analysis of in vitro reconstitutions containing the indicated protein combinations . Coomassie blue - stained SDS - PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms . The red * indicates a contaminant , which we identified by cryo - EM analysis as E . coli ArnA , a notorious contaminant in E . coli Ni - NTA protein purifications ( Andersen et al . , 2013 ) ( Appendix Fig S2 , S3 ) . Note that ArnA is present in GF fraction 6 ( Ve ~ 10 . 2 ml ) of all runs containing E27 , and migrates similarly to E27 on the SDS - PAGE . * - additional contaminant from E27 preparation ; * * - contaminant from STAT2 preparation .

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

Figure 2(A, B) GF analysis of in vitro reconstitutions containing the indicated protein combinations. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms. The red * indicates a contaminant, which we identified by cryo-EM analysis as E. coli ArnA, a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013) (Appendix Fig S2, S3). Note that ArnA is present in GF fraction 6 (Ve ~ 10.2 ml) of all runs containing E27, and migrates similarly to E27 on the SDS-PAGE. * - additional contaminant from E27 preparation; ** - contaminant from STAT2 preparation.


Figure 4 ( E ) Pre - formed DDB1 / DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF . SDS - PAGE analyses of GF fractions are shown next to the chromatogram . The red * indicates the ArnA contaminant ( see Fig 2 ) . * - additional contaminant from E27 preparation .

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

Figure 4(E) Pre-formed DDB1/DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF. SDS-PAGE analyses of GF fractions are shown next to the chromatogram. The red * indicates the ArnA contaminant (see Fig 2). * - additional contaminant from E27 preparation.


Figure 7 ( B ) 293T cells were transfected with expression plasmids encoding E27 - HA or indicated variants . 24 h after transfection , cells were lysed and E27 - HA was precipitated using an HA - specific antibody . Precipitates were separated by SDS - PAGE , blotted , and probed using the indicated antibodies . ( E ) 293T cells were transfected with expression plasmids encoding M27 - FLAG or the indicated M27 - FLAG variants . 24 h after transfection , cells were lysed and M27 - FLAG was precipitated using a FLAG - specific antibody . Precipitates were separated by SDS - PAGE , blotted and probed using the indicated antibodies . ( F ) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants . At the indicated time points , cells were lysed , proteins were separated by SDS - PAGE and blotted . Blots were probed using the indicated antibodies . pIE1 and GAPDH were used as infection and loading controls . ( G ) NIH3T3 reporter cells stably expressing ISRE - Luc were infected with the indicated MCMV strains , treated with IFNβ or IFNλ , and luciferase activity was measured . The error bars represent the standard deviation , N = 4 biological replicates , * * * - P ≤ 0 . 001 ( ANOVA ) . ( H ) BALB / c mice were infected i . p . with WT - MCMV and indicated MCMV mutants . At 3 and 21 d p . i . , salivary glands were collected and frozen . Virus titres were determined from organ homogenates by plaque titration . Titrations were performed in quadruplicates . Bars depict the geometric mean , dots show titres of individual mice ( N = 5 biological replicates ) . DL - detection limit .

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 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 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 7(B) 293T cells were transfected with expression plasmids encoding E27-HA or indicated variants. 24 h after transfection, cells were lysed and E27-HA was precipitated using an HA-specific antibody. Precipitates were separated by SDS-PAGE, blotted, and probed using the indicated antibodies.(E) 293T cells were transfected with expression plasmids encoding M27-FLAG or the indicated M27-FLAG variants. 24 h after transfection, cells were lysed and M27-FLAG was precipitated using a FLAG-specific antibody. Precipitates were separated by SDS-PAGE, blotted and probed using the indicated antibodies.(F) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants. At the indicated time points, cells were lysed, proteins were separated by SDS-PAGE and blotted. Blots were probed using the indicated antibodies. pIE1 and GAPDH were used as infection and loading controls.(G) NIH3T3 reporter cells stably expressing ISRE-Luc were infected with the indicated MCMV strains, treated with IFNβ or IFNλ, and luciferase activity was measured. The error bars represent the standard deviation, N=4 biological replicates, *** - P ≤ 0.001 (ANOVA).(H) BALB/c mice were infected i.p. with WT-MCMV and indicated MCMV mutants. At 3 and 21 d p.i., salivary glands were collected and frozen. Virus titres were determined from organ homogenates by plaque titration. Titrations were performed in quadruplicates. Bars depict the geometric mean, dots show titres of individual mice (N=5 biological replicates). DL - detection limit.


Figure 1B . Structured Illumination Microscopy ( SIM ) images of S . aureus cells labelled with the membrane dye Nile Red . White arrowheads point to tetrads of cells that result from deficient cell splitting . Scale bars , 1μm . D . SIM images of 8325 - 4 ∆ sle1 and 8325 - 4 ∆ ftsK S . aureus cells incubated with purified HisTag - Sle1 - SP . External addition of the protein split the tetrads observed in sle1 and ftsK knock - out strains . Scale bars , 1μm .

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

Figure 1B. Structured Illumination Microscopy (SIM) images of S. aureus cells labelled with the membrane dye Nile Red. White arrowheads point to tetrads of cells that result from deficient cell splitting. Scale bars, 1μm.D. SIM images of 8325-4∆sle1 and 8325-4∆ftsK S. aureus cells incubated with purified HisTag-Sle1-SP. External addition of the protein split the tetrads observed in sle1 and ftsK knock-out strains. Scale bars, 1μm.


Figure 2A . Absence of Sle1 in ( from top to bottom ) autolytic , cell wall or total cellular extracts and spent medium of 8325 - 4 ∆ sle1 and 8325 - 4 ∆ ftsK mutants , but not in the parental strain NCTC8325 - 4 , was confirmed by western blot analysis using an anti - Sle1 antibody . All western blot experiments were performed with three biological replicates and representative images are shown . B . Quantification of Sle1 total cellular levels , detected by western blot ( representative gel on the bottom ) in S . aureus mutant 8325 - 4 ∆ ftsK and ClpX inactivated mutants 8325 - 4 ClpXR95C and 8325 - 4 ∆ ftsK ClpXR95C showing that ClpX inactivation restores the presence of Sle1 in the ftsK deletion mutant . Data information : Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325 - 4 indicated by dashed line . Data represented in scatter dot plot column graphs , where column height represents mean , and error bars are the standard deviation . Data from at least four biological replicates . C . Quantification of Sle1 total cellular levels , detected by western blot ( representative gel on the bottom ) in FtsK C - terminal deletion ( 8325 - 4 ∆ ftsKc ) or inactivated ( 8325 - 4FtsKK971A ) mutants showing that Sle1 levels increase in the absence of FtsK DNA translocase activity . Data information : Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325 - 4 indicated by dashed line . Data represented in scatter dot plot column graphs , where column height represents mean , and error bars are the standard deviation . Data from at least four biological replicates .

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 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

Figure 2A. Absence of Sle1 in (from top to bottom) autolytic, cell wall or total cellular extracts and spent medium of 8325-4∆sle1 and 8325-4∆ftsK mutants, but not in the parental strain NCTC8325-4, was confirmed by western blot analysis using an anti-Sle1 antibody. All western blot experiments were performed with three biological replicates and representative images are shown.B. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom) in S. aureus mutant 8325-4∆ftsK and ClpX inactivated mutants 8325-4 ClpXR95C and 8325-4∆ftsK ClpXR95C showing that ClpX inactivation restores the presence of Sle1 in the ftsK deletion mutant. Data information: Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from at least four biological replicates.C. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom) in FtsK C-terminal deletion (8325-4∆ftsKc) or inactivated (8325-4FtsKK971A) mutants showing that Sle1 levels increase in the absence of FtsK DNA translocase activity. Data information: Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from at least four biological replicates.


Figure 3A . Total protein extracts of strains NCTC8325 - 4 , 8325 - 4 TF - GFP , 8325 - 4 MurC - GFP and 8325 - 4 TF - GFP Sle1Tn ( the latter lacking Sle1 ) were immunoprecipitated with GFP - Trap Magnetic Particles . Western blot analysis of the pulled - downed fractions using antibodies against Sle1 and GFP shows that Sle1 is pulled down with TF - GFP but not with negative control MurC - GFP , suggesting a specific interaction between TF and Sle1 . B . Total protein extracts of strains 8325 - 4 TF - 3XFLAG , 8325 - 4 GFP - FtsK TF - 3XFLAG , 8325 - 4sfgfp - ftsK and 8325 - 4 TF - 3XFLAG GFP - HU ( Input extracts , bottom panel ) were immunoprecipitated with DYKDDDDK Fab - Trap Agarose that binds FLAG - tagged proteins . Western blot analysis of the pulled - downed fractions ( top panels ) using antibodies against FLAG and GFP shows that GFP - FtsK , but not the negative control GFP - HU , is pulled down with TF - 3XFLAG , suggesting an interaction between TF and FtsK . For each assay , a representative image of three independent biological replicates is shown .

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 O O O O O O 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 3A. Total protein extracts of strains NCTC8325-4, 8325-4 TF-GFP, 8325-4 MurC-GFP and 8325-4 TF-GFP Sle1Tn (the latter lacking Sle1) were immunoprecipitated with GFP-Trap Magnetic Particles. Western blot analysis of the pulled-downed fractions using antibodies against Sle1 and GFP shows that Sle1 is pulled down with TF-GFP but not with negative control MurC-GFP, suggesting a specific interaction between TF and Sle1.B. Total protein extracts of strains 8325-4 TF-3XFLAG, 8325-4 GFP-FtsK TF-3XFLAG, 8325-4sfgfp-ftsK and 8325-4 TF-3XFLAG GFP-HU (Input extracts, bottom panel) were immunoprecipitated with DYKDDDDK Fab-Trap Agarose that binds FLAG-tagged proteins. Western blot analysis of the pulled-downed fractions (top panels) using antibodies against FLAG and GFP shows that GFP-FtsK, but not the negative control GFP-HU, is pulled down with TF-3XFLAG, suggesting an interaction between TF and FtsK. For each assay, a representative image of three independent biological replicates is shown.


Figure 4A . SIM images of cells expressing TF - GFP labeled with membrane dye FM5 - 95 . TF - GFP forms a gradient towards the septum in the background of parental strain NCTC8325 - 4 ( 8325 - 4 TF - GFP , top panels ) . This localization pattern is lost in cells lacking FtsK ( 8325 - 4 ∆ ftsK TF - GFP , middle panels ) , but not in 8325 - 4 ∆ ftsKc TF - GFP mutant , expressing FtsK without its C - terminal domain ( bottom panels ) . Scale bars , 1 µm .

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

Figure 4A. SIM images of cells expressing TF-GFP labeled with membrane dye FM5-95. TF-GFP forms a gradient towards the septum in the background of parental strain NCTC8325-4 (8325-4 TF-GFP, top panels). This localization pattern is lost in cells lacking FtsK (8325-4∆ftsK TF-GFP, middle panels), but not in 8325-4∆ftsKc TF-GFP mutant, expressing FtsK without its C-terminal domain (bottom panels). Scale bars, 1 µm.


Figure 5B . Deconvolved epifluorescence images of live cells of parental strain 8325 - 4 Sle1 - 3XFLAG ∆ spa and TF deletion mutant 8325 - 4 Sle1 - 3XFLAG ∆ spa ∆ tig , producing Sle1 - 3XFLAG as the sole Sle1 copy in the cell . Normal localization pattern of Sle1 ( enriched at midcell , top panel ) is altered in the absence of TF ( bottom panel ) with the protein becoming more dispersed over the cell surface . See image S1 for more images of Sle1 - 3XFLAG surface localization in the parental strain and the negative control of the immunofluorescence assay . Cells were incubated with fluorescent D - amino acid HADA , which is incorporated into peptidoglycan , for cell wall visualization ( colored blue ) . Cell surface Sle1 - 3XFLAG was detected with Anti - FLAG Monoclonal Antibody - Alexa Fluor 488 ( colored green , projection of a 33 slices Z - stack shown ) . Scale bars , 1 µm . D . Frequency of cells in each cell cycle phase in the TF knockout mutant 8325 - 4Δtig and NCTC8325 - 4 parental strain , showing that the TF mutant is delayed in cell cycle phase 3 , taking longer to split the division septum . Data from three biological replicates of NCTC8325 - 4 ( Ntotal = 728 ) and 8325 - 4Δtig ( Ntotal = 699 ) . Data represented in column graphs where column height represents mean and error bars are the standard deviation .

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 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

Figure 5B. Deconvolved epifluorescence images of live cells of parental strain 8325-4 Sle1-3XFLAG ∆spa and TF deletion mutant 8325-4 Sle1-3XFLAG ∆spa ∆tig, producing Sle1-3XFLAG as the sole Sle1 copy in the cell. Normal localization pattern of Sle1 (enriched at midcell, top panel) is altered in the absence of TF (bottom panel) with the protein becoming more dispersed over the cell surface. See image S1 for more images of Sle1-3XFLAG surface localization in the parental strain and the negative control of the immunofluorescence assay. Cells were incubated with fluorescent D-amino acid HADA, which is incorporated into peptidoglycan, for cell wall visualization (colored blue). Cell surface Sle1-3XFLAG was detected with Anti-FLAG Monoclonal Antibody-Alexa Fluor 488 (colored green, projection of a 33 slices Z-stack shown). Scale bars, 1 µm.D. Frequency of cells in each cell cycle phase in the TF knockout mutant 8325-4Δtig and NCTC8325-4 parental strain, showing that the TF mutant is delayed in cell cycle phase 3, taking longer to split the division septum. Data from three biological replicates of NCTC8325-4 (Ntotal=728) and 8325-4Δtig (Ntotal=699). Data represented in column graphs where column height represents mean and error bars are the standard deviation.


Figure 6A . Quantification of Sle1 total cellular levels , detected by western blot ( representative gel on the bottom ) , in S . aureus cultures of wild - type strain NCTC8325 - 4 grown in the presence ( 1 / 2 X MIC ) or absence of DNA targeting antibiotics mitomycin C and nalidixic acid or protein synthesis inhibitor erythromycin . The results show a decrease in Sle1 cellular levels upon DNA replication defects or DNA damage . Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325 - 4 indicated by dashed line . Data represented in scatter dot plot column graphs , where column height represents mean , and error bars are the standard deviation . Data from four biological replicates . Statistical significance based on a Mann - Whitney U test , where * p < 0 . 05 . C . SIM image and heat map representation of TF - GFP localization in 8325 - 4 TF - GFP cells grown in the presence or absence of nalidixic acid , showing the loss of TF - GFP gradient towards the septum in most cells grown in the presence of the antibiotic . Scale bars , 1 µm . Heatmaps represent the average of TF - GFP localization in 50 cells in phase 2 of the cell cycle . The color scale depicts the average normalized TF - GFP fluorescence signal in each pixel , ranging between 0 and 1 . The fraction of the area of each cell model where the normalized signal intensity is above 70 % of the maximum signal is 0 . 47 and 0 . 61 in the absence or presence of nalidixic acid , respectively , confirming that TF - GFP becomes more dispersed over the cytoplasm in the presence of the antibiotic .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. Quantification of Sle1 total cellular levels, detected by western blot (representative gel on the bottom), in S. aureus cultures of wild-type strain NCTC8325-4 grown in the presence (1/2 X MIC) or absence of DNA targeting antibiotics mitomycin C and nalidixic acid or protein synthesis inhibitor erythromycin. The results show a decrease in Sle1 cellular levels upon DNA replication defects or DNA damage. Graphs show ratio between Sle1 amount in different samples versus the amount in parental strain NCTC8325-4 indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from four biological replicates. Statistical significance based on a Mann-Whitney U test, where * p< 0.05.C. SIM image and heat map representation of TF-GFP localization in 8325-4 TF-GFP cells grown in the presence or absence of nalidixic acid, showing the loss of TF-GFP gradient towards the septum in most cells grown in the presence of the antibiotic. Scale bars, 1 µm. Heatmaps represent the average of TF-GFP localization in 50 cells in phase 2 of the cell cycle. The color scale depicts the average normalized TF-GFP fluorescence signal in each pixel, ranging between 0 and 1. The fraction of the area of each cell model where the normalized signal intensity is above 70% of the maximum signal is 0.47 and 0.61 in the absence or presence of nalidixic acid, respectively, confirming that TF-GFP becomes more dispersed over the cytoplasm in the presence of the antibiotic.


Figure 3A Example of the mapping results . Wild allele frequency in progeny of a cross of the gab1 - 1 temperature sensitive allele to the YJM975 strain ( y - axis ) along the yeast genome ( x - axis ) at the permissive 26 ° C ( blue ) and restrictive 34 ° C ( TS allele loss - of - function , cyan ) . The allele frequency change between the two temperatures is used in mapping . Labels : selected loci in the cross . Blue regions : called suppressor loci . B

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

Figure 3A Example of the mapping results. Wild allele frequency in progeny of a cross of the gab1-1 temperature sensitive allele to the YJM975 strain (y-axis) along the yeast genome (x-axis) at the permissive 26°C (blue) and restrictive 34°C (TS allele loss-of-function, cyan). The allele frequency change between the two temperatures is used in mapping. Labels: selected loci in the cross. Blue regions: called suppressor loci. B


Figure 4E Experimental validation of RPN8 as the causal suppressor of the rpn11 - 8 TS mutant . Cultures of the indicated strains were diluted to an optical density at 600 nm of 0 . 1 and a series of ten - fold dilutions was spotted on agar plates and incubated for 2 - 3 days at 34 ° C . UWOPS = UWOPS87 - 2421 . See also Figure EV4 .

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

Figure 4E Experimental validation of RPN8 as the causal suppressor of the rpn11-8 TS mutant. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days at 34°C. UWOPS = UWOPS87-2421. See also Figure EV4.


Figure 5A , B Suppression of nse3 - ts4 and nse4 - ts4 temperature sensitivity ( A ) and DNA damage sensitivity ( B ) by the NSE1 allele of UWOPS87 - 2421 . Cultures of the indicated strains were diluted to an optical density at 600 nm of 0 . 1 and a series of ten - fold dilutions was spotted on agar plates and incubated for 2 - 3 days . UW = UWOPS87 - 2421 . The plates shown in ( B ) were incubated at 30 ° C . C E Recruitment of Smc6 - FLAG by ChIP - qPCR at two known SMC5 / 6 binding sites ( pericentromere of chromosome XIV and CEN3 ) and one negative control locus ( arm of chromosome III ) in G2 / M arrested strains . Relative enrichment corresponds to the ratio of the signal after immunoprecipitation ( FLAG ) over beads alone , normalised to the ratio in wild - type cells at CEN3 . Error bars : standard error of the mean of three independent experiments . Statistical significance was determined using an unpaired , two - tailed t - test ( * = p < 0 . 05 , * * = p < 0 . 005 ) . E

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 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

Figure 5A,B Suppression of nse3-ts4 and nse4-ts4 temperature sensitivity (A) and DNA damage sensitivity (B) by the NSE1 allele of UWOPS87-2421. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days. UW = UWOPS87-2421. The plates shown in (B) were incubated at 30°C. C E Recruitment of Smc6-FLAG by ChIP-qPCR at two known SMC5/6 binding sites (pericentromere of chromosome XIV and CEN3) and one negative control locus (arm of chromosome III) in G2/M arrested strains. Relative enrichment corresponds to the ratio of the signal after immunoprecipitation (FLAG) over beads alone, normalised to the ratio in wild-type cells at CEN3. Error bars: standard error of the mean of three independent experiments. Statistical significance was determined using an unpaired, two-tailed t-test (* = p < 0.05, ** = p < 0.005). E


Figure 1 ( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test . ( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the hepatocytes expressing GFP . The experiments were performed three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test . ( C ) Immunofluorescence microscopy . Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test . ( D ) FRAP assay . Wild type hepatocytes cultured in glass - bottom plates were infected with GFP - NBR1 adenovirus for 48 hrs . The signal recovery after photobleaching was measured , quantified from at least three independent biological replicates , and is represented as means ± s . e . Bar : 1 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 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 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 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

Figure 1(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.(C) Immunofluorescence microscopy. Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.(D) FRAP assay. Wild type hepatocytes cultured in glass-bottom plates were infected with GFP-NBR1 adenovirus for 48 hrs. The signal recovery after photobleaching was measured, quantified from at least three independent biological replicates, and is represented as means ± s.e. Bar: 1 µm.


Figure 2 ( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with adenovirus expressing GFP - Nrf2 and GFP or NBR1 for 48 hrs . Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * * P < 0 . 01 as determined by Welch ' s t - test . ( B ) Immunoblot analysis . Primary hepatocytes prepared from p62f / f and p62f / f ; Alb - Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs . Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin ( cytosolic proteins ) or Lamin B ( nuclear proteins ) . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test . ( C ) Immunofluorescence microscopy . Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP - Nrf2 and LacZ or NBR1 for 48 hrs . More than 100 cells were assayed for the nuclear signal of Nrf2 . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test . Bars : 10 µm . ( D ) Total RNAs were prepared from primary hepatocytes described in ( B ) . Values were normalized against the amount of mRNA in the p62f / f hepatocytes expressing GFP . The experiments were performed at least three times . Data are are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' 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 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 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 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 2(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP-Nrf2 and GFP or NBR1 for 48 hrs. Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. **P < 0.01 as determined by Welch's t-test.(B) Immunoblot analysis. Primary hepatocytes prepared from p62f/f and p62f/f; Alb-Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.(C) Immunofluorescence microscopy. Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP-Nrf2 and LacZ or NBR1 for 48 hrs. More than 100 cells were assayed for the nuclear signal of Nrf2. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test. Bars: 10 µm.(D) Total RNAs were prepared from primary hepatocytes described in (B). Values were normalized against the amount of mRNA in the p62f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


Figure 3 ( A ) Immunofluorescence microscopy . Primary hepatocytes prepared from p62f / f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1 . 24 hrs after the infection , the hepatocytes were immunostained with anti - Keap1 , anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 10 µm . ( B ) Immunoprecipitation analysis . Crude extracts were prepared from primary hepatocytes as described in ( A ) and then immunoprecipitated with anti - FLAG ( as a negative control ) or anti - NBR1 antibody . The resulting samples were analyzed by immunoblot analysis . Data shown are representative of three separate experiments . ( C ) Primary hepatocytes prepared from p62f / f ; Alb - Cre mice were co - infected with adenovirus GFP or NBR1 in combination with wild - type p62 or the mutants for 48 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' s t - test . ( D ) RT qPCR analysis . Total RNAs were prepared from hepatocytes described in ( C ) . Values were normalized against the amount of mRNA in the hepatocytes expressing GFP . The experiments were performed three times . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' 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 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 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

Figure 3(A) Immunofluorescence microscopy. Primary hepatocytes prepared from p62f/f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1. 24 hrs after the infection, the hepatocytes were immunostained with anti-Keap1, anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 10 µm.(B) Immunoprecipitation analysis. Crude extracts were prepared from primary hepatocytes as described in (A) and then immunoprecipitated with anti-FLAG (as a negative control) or anti-NBR1 antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three separate experiments.(C) Primary hepatocytes prepared from p62f/f; Alb-Cre mice were co-infected with adenovirus GFP or NBR1 in combination with wild-type p62 or the mutants for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.(D) RT qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


Figure 4 ( A ) Immunoblot analysis . Primary hepatocytes prepared from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs . Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin ( cytosolic proteins ) or Lamin B ( nuclear proteins ) . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - test . ( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the Nrf2f / f hepatocytes expressing GFP . The experiments were performed at least three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test . ( C ) Immunofluorescence microscopy . Hepatocytes isolated from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' 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 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

Figure 4(A) Immunoblot analysis. Primary hepatocytes prepared from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nrf2f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.(C) Immunofluorescence microscopy. Hepatocytes isolated from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


Figure 5 ( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 ( BafA1 ) for 24 hrs or cultured in amino acid - deprived medium ( Stv ) for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test . ( B ) Fluorescence microscopy . p62 - deficient MEFs were co - transfected with mCherry - GFP - p62 in combination with wild - type NBR1 or NBR1 D50R mutant . 48 hrs after the transfection , fluorescence images were observed . Bars : 10 µm . The number of puncta positive for only red - signal in each MEFs was counted in more than 100 cells . Bar graphs are represented as means ± s . e . * * * P < 0 . 001 as determined by Welch ' 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 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

Figure 5(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 (BafA1) for 24 hrs or cultured in amino acid-deprived medium (Stv) for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.(B) Fluorescence microscopy. p62-deficient MEFs were co-transfected with mCherry-GFP-p62 in combination with wild-type NBR1 or NBR1 D50R mutant. 48 hrs after the transfection, fluorescence images were observed. Bars: 10 µm. The number of puncta positive for only red-signal in each MEFs was counted in more than 100 cells. Bar graphs are represented as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


Figure 6 ( A ) Immunoblot analysis . Primary hepatocytes were isolated from wild - type ( wt ) , p62f / f ; Alb - Cre ( p62KO ) and Nrf2f / f ; Alb - Cre ( Nrf2KO ) mice . The hepatocytes were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with anti - NBR1 and anti - Actin antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - test . ( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in each non - treated hepatocytes . The experiments were performed three times . Data are shown as means ± s . e . * * P < 0 . 01 and * * * P < 0 . 001 as determined by Welch ' s t - test . ( C ) Immunofluorescence microscopy . Primary hepatocytes isolated from wild - type mice were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' 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 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 O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes were isolated from wild-type (wt), p62f/f; Alb-Cre (p62KO) and Nrf2f/f; Alb-Cre (Nrf2KO) mice. The hepatocytes were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with anti-NBR1 and anti-Actin antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in each non-treated hepatocytes. The experiments were performed three times. Data are shown as means ± s.e. **P < 0.01 and ***P < 0.001 as determined by Welch's t-test.(C) Immunofluorescence microscopy. Primary hepatocytes isolated from wild-type mice were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.