C of centrosome numbers ( marked by γ - tubulin ) upon CB treatment from ( B ) . For left panel , in total 632 ( untx , T24 ) , 637 ( CB , T24 ) , 544 ( untx , T48 ) 467 ( CB , T48 ) from two litters were analyzed . T24 = 24 hours . T48 = 48 hours . For right panel , in total 799 ( untx , non - enucl ) , 142 ( untx , enucl ) , 235 ( untx , RetiC ) , 839 ( CB , non - enucl ) , 127 ( CB , enucl ) 131 ( CB , RetiC ) from two litters were analysed . Data information : Bar graphs display mean ± s . d . Statistical was on number of litters ( C * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Quantification of centrosome numbers (marked by γ-tubulin) upon CB treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunofluorescence of untreated ( untx ) or centrinone - B ( CB ) - treated at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , phospho - Histone H3 ( pHH3 , green ) ( Hoechst , blue ) . are maximum projections of deconvolved z - stacks . Scale bar , 2 μm . B of mitotic spindle morphologies in untreated ( untx ) or centrinone - B ( CB ) - treated at T24 or T36 of ex vivo culture . Graph depicts of spindle . In total 315 ( untx , T24 ) , 367 ( CB , T24 ) , 295 ( untx , T36 ) , 282 ( CB , T36 ) were analyzed from 3 litters . Data information : Bar graphs display mean ± s . d . Statistical was on number of litters ( B ) Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons ( B , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Immunofluorescence images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


C Immunofluorescence of Cdk5rap2HET Cdk5rap2null at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , pHH3 ( green ) ( Hoechst , blue ) . are maximum projections of deconvolved z - stacks . Scale bar , 2 μm . D of mitotic spindle at T24 or T36 of ex vivo culture . Graph depicts of spindle . At T24 , 3 Cdk5rap2WT ( 323 ) , 3 Cdk5rap2HET ( 296 ) 2 Cdk5rap2null ( 197 ) were analyzed . At T36 , 5 Cdk5rap2WT ( 387 ) , 6 Cdk5rap2HET ( 384 ) 3 Cdk5rap2null ( 202 ) were analyzed . Data information : Bar graphs display mean ± s . d . Statistical was on number of ( D , Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons D , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), pHH3 (green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, 3 Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2 Cdk5rap2null (197 cells) embryos were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (D, Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test D, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


E Schematic ( left ) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points . Representative ImageStream of ex vivo cultured at different mitotic stages ( right ) . were stained for phospho - Histone H3 ( pHH3 , green ) , TER119 ( erythroid marker , magenta ) ( Hoechst , blue ) . BF : brightfield . Scale bar , 10 μm . F G of pHH3pos , TER119pos mitotic stages ( see text for details ) after 36 hours ( T36 ) upon DMSO or nocodazole treatment according to ( E ) using ImageStream . Number of analyzed is shown in brackets . Data information : Box plots show 5th , 95th ( whiskers ) 25th , 50th 75th percentile ( boxes ) . Statistical was on number of F G ) . Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons F G ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

E Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho-Histone H3 (pHH3, green), TER119 (erythroid marker, magenta) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 10 μm. F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


B Immunofluorescence of in interphase ( I ) mitosis ( M ) at 24 ( T24 ) 36 ( T36 ) hours of ex vivo culture . were stained for γ - tubulin ( magenta ) , of interest ( POI , CDK5RAP2 , PCNT or CEP192 in grey ) ( Hoechst , blue ) . are maximum projections of deconvolved z - stacks . Scale bar , 2 μm . C of mean centrosomal signal intensities of PCM from ( B ) . Three litters were analyzed with following number of for I , T24 ; M , T24 ; I , T36 M , T36 : 290 , 157 , 259 174 ( CDK5RAP2 ) ; 291 , 190 , 203 181 ( PCNT ) ; 314 , 122 , 283 152 ( CEP192 ) ; 895 , 465 , 720 493 ( γ - tubulin ) . Data information : Bar graphs display mean ± s . d . Statistical was on number of litters ( C Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

B Immunofluorescence images of cells in interphase (I) and mitosis (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT or CEP192 in grey) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. C Quantification of mean centrosomal signal intensities of PCM proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin). Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunoblot of TP53 , P27 P21 in ex vivo cultured Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) null EBs after 24 ( T24 ) 48 ( T48 ) hours . GAPDH was used as loading control . * * indicates unspecific band . B of mean from ( A ) . Numbers in brackets represent number of analyzed . Data information : Box plots show 5th , 95th ( whiskers ) 25th , 50th 75th percentile ( boxes ) . statistical were on number of . statistical significances were determined by One - way ANOVA with Tukey ' s multiple comparisons . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Immunoblot analysis of TP53, P27 and P21 levels in ex vivo cultured Cdk5rap2 wild-type (WT), heterozygous (HET) and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** indicates unspecific band. B Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). All statistical analysis were based on number of embryos. All statistical significances were determined by One-way ANOVA with Tukey's multiple comparisons test.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A . Expression of various GPI - APs in different clones of PIGG - KO HEK293 . intensities of NT5E in PIGG - KO clones # 4 , # 10 , # 17 , # 19 wild type are 575 , 910 , 696 , 530 1548 while those of EphrinA5 in clones # 4 , # 17 , # 19 wild type are 1431 , 2080 , 1868 3336 , respectively . Data information : Each FACS was performed at least three times .

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

A. Expression levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively. Data information: Each FACS analysis was performed at least three times.


B . Expression of GPI - APs in PIGG - KO clone # 19 stably transfected with PIGG cDNA compared to in empty vector transfected . intensities of NT5E in PIGG cDNA in vector transfected are 1332 886 , respectively . Data information : Each FACS was performed at least three times .

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

B. Expression levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells. Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886, respectively. Data information: Each FACS analysis was performed at least three times.


A . Expression of different GPI - APs in PIGG - KO HEK293 transiently co - transfected with PIGG cDNA or empty vector . Vertical bars represent % of of GPI - APs expression in non - rescued PIGG - KO relative to those in PIGG cDNA rescued PIGG - KO . was repeated 3 to 6 times SD bars were shown .

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

A. Expression levels of different GPI-APs in PIGG-KO HEK293 cells transiently co-transfected with PIGG cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.


B . HA tagged NTNG2 FLAG tagged DAF were transiently co - transfected together with PIGG cDNA or empty vector to PIGG - KO were analyzed by FACS . of NTNG2 in PIGG - KO in PIGG rescued are 639 3426 , while those of DAF in PIGG - KO in PIGG rescued are 562 599 , respectively . Each FACS was performed at least three times .

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

B. HA tagged NTNG2 and FLAG tagged DAF were transiently co-transfected together with PIGG cDNA or empty vector to PIGG-KO cells and were analyzed by FACS. Fluorescence intensity levels of NTNG2 in PIGG-KO cells and in PIGG rescued cells are 639 and 3426, while those of DAF in PIGG-KO cells and in PIGG rescued cells are 562 and 599, respectively. Each FACS analysis was performed at least three times.


( A ) IP of GFP - Atg8 ( α ‐ GFP ) , Atg30 - Flag ( α ‐ Flag ) Pex3 ( α ‐ Pex3 ) under pexophagy conditions . abundant peroxisome matrix , AOX , was used as a negative control . Input : total lysate ; ϕ : IP without antibody .

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

(A) IP of GFP-Atg8 (α‐GFP), Atg30-Flag (α‐Flag) and Pex3 (α‐Pex3) under pexophagy conditions. The abundant peroxisome matrix protein, AOX, was used as a negative control. Input: total lysate; ϕ: IP without antibody.


( B ) Two multiple alignments , including 11 Atg30 homologues 11 Atg32 homologues ( identical residues are indicated with black boxes , similar residues with grey boxes ) , a logo of combined multiple alignments from Atg30 Atg32 homologues listed currently in GenBank .

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

(B) Two multiple sequence alignments, including 11 Atg30 homologues and 11 Atg32 homologues (identical residues are indicated with black boxes, and similar residues with grey boxes), and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank.


( C , D ) Pexophagy experiments of Δatg30 ( ϕ ) , wild ‐ type ( Atg30 ) Atg30 AIM mutant ( Atg30W73A F76A ) were done by microscopy , following degradation of peroxisomes labelled with BFP fused at its C ‐ terminus to Ser - Lys - Leu peroxisomal targeting signal 1 ( BFP - SKL ) biochemically by monitoring peroxisomal thiolase degradation . Vacuoles were labelled with FM4 ‐ 64 . Scale bar , 5 μm .

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

(C,D) Pexophagy experiments of Δatg30 (ϕ), wild‐type (Atg30) and Atg30 AIM mutant (Atg30W73A F76A) cells were done by fluorescence microscopy, following the degradation of peroxisomes labelled with BFP fused at its C‐terminus to the Ser-Lys-Leu peroxisomal targeting signal 1 (BFP-SKL) and biochemically by monitoring peroxisomal thiolase degradation. Vacuoles were labelled with FM4‐64. Scale bar, 5 μm.


( E ) AH109 were transformed with two yeast two ‐ hybrid plasmids , AD BD , which encode indicated domains fused with Atg30 , Atg32 Atg8 or an empty vector , as negative controls grown on + His − His + 40 mM 3 ‐ AT plates .

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

(E) AH109 cells were transformed with two yeast two‐hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3‐AT plates.


( F ) . Δatg30 ( ϕ ) Δatg30 complemented with Atg30 - HA ( Atg30 ) several Atg30 - HA mutants were immunoprecipitated ( α ‐ HA IP ) under pexophagy conditions . In addition , α ‐ HA IP of Δatg30 ( ϕ ) Δatg30 complemented with Atg30 - HA ( Atg30 ) were incubated with ( + ) without ( − ) λPP . Input : total lysate .

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

(F) . Δatg30 (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) and several Atg30-HA mutants were immunoprecipitated (α‐HA IP) under pexophagy conditions. In addition, α‐HA IP of Δatg30 cells (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) were incubated with (+) and without (−) λPP. Input: total lysate.


( G ) Pexophagy in atg30 mutants was monitored by following thiolase of oleate ‐ induced peroxisomes after shifting to SD ‐ N . aa , acid ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; AOX , alcohol oxidase ; BD , binding domain ; GFP , green ; HA , haemagglutinin ; IP , immunoprecipitation ; λPP , λ phosphatase .

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

(G) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate‐induced peroxisomes after shifting cells to SD‐N. aa, amino acid; AD, activation domain; AIM, Atg8‐family‐interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.


( A1 ) Atg30 sequences from aa 68 - 122 of wild ‐ type ( Atg30 ) , S71A mutant ( Atg30S71A ) two different deletions of between AIM phosphosite required for Atg11 binding in Atg30 ( Atg30WDILSSS Atg30WSILSSS ) .

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

(A1) Atg30 sequences from aa 68-122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS).


( A2 ) Pexophagy experiments of Δatg30 complemented with appropriate wild ‐ type or mutant Atg30 described in A1 .

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

(A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1.


( A3 ) Two ‐ hybrid assays between Atg30 wild ‐ type or mutants ( described in A1 ) Atg8 . Phosphomimic S71E was included to detect interaction with Atg8 .

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

(A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8.


( B2 ) Pexophagy experiments of Δatg30 complemented with two Atg30 molecules described in B1 .

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

(B2) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1.


( C2 ) Pexophagy assays of Δatg30 complemented with Atg30 wild ‐ type mutants . aa , ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; HA , haemagglutinin .

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

(C2) Pexophagy assays of Δatg30 cells complemented with Atg30 wild‐type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; HA, haemagglutinin.


( A ) Large phagophore formation in WT Atg30 mutant monitored by GFP - Atg8 during pexophagy conditions .

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

(A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP-Atg8 during pexophagy conditions.


( B ) Localization of GFP - Atg11 during pexophagy of methanol ‐ induced peroxisomes in expressing WT or mutant Atg30 . arrows indicate correct localization yellow arrows indicate mislocalization , or in case of Atg8 localization , indicate absence of phagophore elongation . Peroxisomes were labelled with BFP - SKL vacuoles with FM4 ‐ 64 . Scale bar , 5 μm . GFP , green ; WT , wild type .

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(B) Localization of GFP-Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP-SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.


( A ) Mitophagy experiments of WT ( PPY12 ) , Δatg5 , Δatg32 ( ϕ ) Δatg32 complemented with WT Atg32 ( Atg32 ) , Atg32 with a deletion of between AIM phosphosite required for Atg11 binding ( Atg32WQVLSSS ) , Atg32 AIM mutant ( Atg32W121A V124A ) , mutants of Thr upstream of Atg32 AIM ( Atg32T119A ) mutants altered in Ser required for Atg11 binding ( Atg32S159A ) . were grown in YPL medium shifted to SD ‐ N . Mitophagy was followed by transport of Tom20 - mCherry to vacuole by microscopy . Mitophagy was classified as − no mitophagy , + few show mitophagy , + + + most show mitophagy + + + + almost show mitophagy ( numbers of containing Tom20 - mCherry in vacuole was considered ) . Scale bar , 5 μm .

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(A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32WQVLSSS), Atg32 AIM mutant (Atg32W121A V124A), mutants of the Thr upstream of the Atg32 AIM (Atg32T119A) and mutants altered in the Ser required for Atg11 binding (Atg32S159A). The cells were grown in YPL medium and shifted to SD‐N. Mitophagy was followed by the transport of Tom20-mCherry to the vacuole by fluorescence microscopy. Mitophagy was classified as −no mitophagy,+few cells show mitophagy, +++ most cells show mitophagy and ++++ almost all the cells show mitophagy (the intensity and numbers of cells containing Tom20-mCherry in the vacuole was considered). Scale bar, 5 μm.


( B ) P . pastoris Δatg32 ( ΔPpatg32 ) expressing Tom20 - GFP expressing indicated Atg32 mutants were cultured in YPL medium for 12 , 18 36 h . Mitophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

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(B) P. pastoris Δatg32 cells (ΔPpatg32) expressing Tom20-GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12, 18 and 36 h. Mitophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( C ) N ‐ terminal of Atg36 manually aligned against several alignments of Atg30 , Atg32 ScAtg32 . Identical residues are indicated with black boxes , similar residues with grey boxes .

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(C) N‐terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30, Atg32 and ScAtg32. Identical residues are indicated with black boxes, and similar residues with grey boxes.


( D ) Two ‐ hybrid - interaction of ScAtg36 , ScAtg8 ScAtg11 . receptors were mutated at AIM ( Atg36F33A L36A ) , at serine ( s ) upstream of AIM ( Atg36S31A ) at Atg11 ‐ binding site ( Atg36S97A ) .

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(D) Two‐hybrid protein-protein interaction analysis of ScAtg36, ScAtg8 and ScAtg11. The receptors were mutated at the AIM (Atg36F33A L36A), at serine(s) upstream of the AIM (Atg36S31A) and at the Atg11‐binding site (Atg36S97A).


( E ) S . cerevisiae Δatg36 ( ΔScatg36 ) expressing thiolase - GFP expressing indicated Atg36 mutants were cultured in oleate medium until mid ‐ log growth then shifted to SD ‐ N . Pexophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

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(E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase-GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid‐log growth and then shifted to SD‐N. Pexophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( F ) Y3H of ScAtg36 with ScAtg8 ScAtg11 . Y3H technology is on basis of yeast two ‐ hybrid system but with co ‐ expression of third as a competitor indicated in figure ( NLS - ScAtg11 or NLS - ScAtg8 ) . positive control was ScAtg36 mutant affected in Atg11 binding ( ScAtg36S97A ) or in Atg8 binding ( ScAtg36S31A ) , which should be unaffected by competition of NLS - ScAtg11 or NLS - ScAtg8 , respectively . Appropriate auto ‐ activation interaction controls were also included . AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; GFP , green ; WT , wild type ; Y3H , yeast three ‐ hybrid .

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(F) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11. The Y3H technology is on the basis of the yeast two‐hybrid system but with the co‐expression of third protein as a competitor and indicated in the figure (NLS-ScAtg11 or NLS-ScAtg8). The positive control was the ScAtg36 mutant affected in Atg11 binding (ScAtg36S97A) or in Atg8 binding (ScAtg36S31A), which should be unaffected by the competition of NLS-ScAtg11 or NLS-ScAtg8, respectively. Appropriate auto‐activation and interaction controls were also included. AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; GFP, green fluorescent protein; WT, wild type; Y3H, yeast three‐hybrid.


( A ) 786 - 0 or 769 - P stably expressing shNT or shGDH1 were treated with or without aa starvation for indicated times , then viability was assessed . ( B ) 786 - 0 or 769 - P stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT ( 3 * WT indicates 3 - fold expression WT ) then treated with aa starvation for indicated time . viability was subsequently assessed . ( Data information : data are represented as means ± of three independent experiments statistical analyses were conducted using unpaired t - . ( * * p < 0 . 01 ; * * * p < 0 . 001 . )

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(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed. (B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.)


( I ) of GDH1 in KIRC tumors adjacent normal tissues collected by us was performed ( left ) . Quantitative of GDH1 was performed using unpaired t - ( right ) . * * * p < 0 . 001 . Scale bars : 50 μm .

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(I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm.


( K ) Overall survival of in different stages in TCGA - KIRC cohort . R package " survminer " - maximally selected log - rank statistic was used to determine best cutoff . log - rank was used to determine p value .

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(K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.


( D ) Immunofluorescence ( IF ) was performed to determine subcellular location of GDH1 with or without aa starvation . GDH1 localization was indicated by GDH1 antibody MitoTracker ( left ) . Signal intensities distances were quantified ( right ) . Scale bars : 20 μm .

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(D) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( E ) Cytoplasm - mitochondrial fractionation was performed to determine subcellular location of GDH1 with or without aa starvation . fractionation efficiency GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial marker ) GDH1 . Tubulin was used as loading control .

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(E) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( G ) Immunofluorescence ( IF ) was performed to determine subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . GDH1 localization was indicated by GDH1 antibody MitoTracker ( left ) . Signal intensities distances were quantified ( right ) . Scale bars : 20 μm .

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(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( H ) Cytoplasm - mitochondrial fractionation was performed to determine subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . fractionation efficiency GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial marker ) GDH1 . Tubulin was used as loading control .

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(H) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( D ) FLAG - tagged GDH1 ( including WT mutants with different lysine mutations to arginine ) was co - transfected with HA - tagged ubiquitin for 48 hours , then were subjected to aa starvation for 12 hours before collecting . WB qPCR separately tested of GDH1 mRNA , respectively . Data information : data are represented as mean ± of three independent experiments . one - way ANOVA ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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(D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


( H ) 786 - 0 or 769 - P stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant then treated with or without aa starvation for indicated time . viability was determined . Unpaired two - t - was used . Data information : data are represented as mean ± of three independent experiments . unpaired t - were used ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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(H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


786 - 0 were treated with MG132 ( 10 μM ) under aa starvation . Antibodies against GDH1 ( C ) were used to enrich complexes in with or without GDH1 . IgG was used as a control . WB was performed using indicated antibodies .

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786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.


( H ) 786 - 0 or 769 - P ( WT or stably expressing shGDH1 ) were transfected with siNC or siRNF213 for 48 hours then were treated with or without aa starvation for indicated times . viability was determined . Unpaired t - was used with * * * p < 0 . 001 .

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(H) 786-0 or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired t-test was used with ***p < 0.001.


( I ) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 ( upper panel ) . correlated signal between GDH1 RNF213 in KIRC samples was calculated ( lower panel ) . cor . was used to determine p - value . Scale bars : 50 μm .

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(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.


786 - 0 expressing WT or enzymatically dead ( ED ) GDH1 were transfected with siNC or siRNF213 for 48 hours then were treated with or without aa starvation for 12 hours . In above in ( A ) , qPCR was used to measure mRNA of indicated RP genes ( C ) .

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786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).


( J ) KIRC clinical samples were collected for αKG measurement , we assessed overall survival ( left panel ) with log - rank according to dividing groups of high low intracellular of αKG with best separation ( right panel ) . Unpaired t - was used in right panel with * * * p < 0 . 001 .

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(J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.


( G ) An in vitro demethylation was performed with recombinant KDM4A ( 0 . 05 mM ) or KDM6A ( 0 . 05 mM ) , indicated histone H3 lysine methylation peptides under 50 - μM αKG conditions ( no αKG was used as a blank control ) . Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in reaction system .

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(G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.


( H ) 786 - 0 were supplemented with or without 2HG ( 500 μM ) for 48 hours , were fixed with 1 % formaldehyde . ChIP assays were performed using antibodies against H3K9me3 or H3K27me3 . qPCR was used to enrichment of above histone H3 lysine modifications on promoters of indicated RP genes .

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(H) 786-0 cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.


Xenogeneic inoculation of 786 - 0 lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into left groin of nude mice . solid formed by indicated 786 - 0 is shown ( B ) .

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Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B).


Xenogeneic inoculation of 786 - 0 lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into left groin of nude mice . of Ki67 ( G , upper panel ) TUNEL ( G , lower panel ) using tumors from ( B ) are shown analyzed ( G , right panel ) . We used 6 mice in each group . Scale bars : 50 μm . Data information : statistical analyses were conducted using unpaired t - ( n = 6 ) . ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


heat map shows position ( left to right ) , identity ( top row ) frequency ( color code ) of variant nucleotide positions detected by SARS - CoV - 2 full genome amplicon sequencing in ( A ) 20 samples of MPP - R workers tested positive on May 20 or 25 , ( B ) 15 samples of MPP - R workers tested positive between June 5 17 , ( C ) two workers from MPP - D who may have transmitted to cases B1 / or B2 . collection dates are shown to right of each sample . Variant sequences are given relative to Wuhan reference NC _ 045512 . two silent mutations which define prototype of investigated outbreak are marked with an asterisk . Frequencies below 100 % mean that only a fraction of viral genomes shows nucleotide variations , indicating presence of viral intra - host sub - populations . rectangles denote nucleotide positions which were not covered by amplicon - seq reads within respective sample . For nucleotide positions in coding regions , corresponding viral ORF ( s ) are shown above variant position . Variants without such information are located in non - coding regions .

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The heat map shows the position (left to right), identity (top row) and frequency (color code) of variant nucleotide positions detected by SARS-CoV-2 full genome amplicon sequencing in (A) 20 samples of MPP-R workers tested positive on May 20 or 25, (B) 15 samples of MPP-R workers tested positive between June 5 and 17, and (C) two workers from MPP-D who may have transmitted the virus to cases B1 and/or B2. Individual collection dates are shown to the right of each sample. Variant sequences are given relative to the Wuhan reference strain NC_045512. The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk. Frequencies below 100% mean that only a fraction of viral genomes shows nucleotide variations, indicating the presence of viral intra-host sub-populations. White rectangles denote nucleotide positions which were not covered by amplicon-seq reads within the respective sample. For nucleotide positions in coding regions, the corresponding viral ORF(s) are shown above the variant position. Variants without such information are located in non-coding regions.


( A ) ( in meters ) of PCR - tested workers from suspected index case B1 at workplace . For workers without fixed position in beef processing ( marked with an asterisk ) coordinates indicate estimated average location during early shift . Squares diamonds denote prototype or variant SARS - CoV - 2 genotypes , respectively . Filled blue circles denote cases for which viral genomes were not sequenced ( i . e . , workers tested positive after 5 / 25 / 2020 ) .

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(A) Distance (in meters) of PCR-tested workers from the suspected index case B1 at the workplace. For workers without fixed position in the beef processing plant (marked with an asterisk) coordinates indicate estimated average location during the early shift. Squares and diamonds denote prototype or variant SARS-CoV-2 genotypes, respectively. Filled blue circles denote cases for which viral genomes were not sequenced (i.e., workers tested positive after 5/25/2020).


( B ) Top panel : Observed accumulated of positive cases ( red ) within indicated from suspected index case . gray dashed shows average infection rate that would be expected for a random spatial distribution of positive cases . Bottom panel : - log10 p - value for frequency of accumulated positive cases within given being significantly higher than expected on a random spatial distribution of positive cases Only employees with fixed work positions were included in calculation .

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(B) Top panel: Observed accumulated percentage of positive cases (red line) within the indicated distance from the suspected index case. The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases. Bottom panel: -log10 p-value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation.


HEK293T were transfected for 48 hours with each mitoTev - TALE ( 11 . 5 8 . 5 RVDs ) total extracts were used for western - blots with anti - T2A antibody . Molecular weights of two different mitoTev - TALEs are ~ 85KD ~ 70KDa ,

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HEK293T cells were transfected for 48 hours with each mitoTev-TALE (11.5 and 8.5 RVDs) and total cell extracts were used for western-blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ~85KD and ~70KDa, respectively


Immunocytochemistry using same antibody along with Mitotracker red was performed in COS7 , 24 hours after transfection . Nuclei were counterstained with DAPI ( blue ) 11 . 5RVD 8 . 5RVD mitoTev - TALEs both co - localized with mitochondria , as seen in merge image . Scale bar : 10

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Immunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm


MtDNA heteroplasmy analyzed by PCR / RFLP , 24 hours post mitoTev - TALE mitoTALEN transfection . RFLP shows increased % WT mtDNA in sorted when compared to untransfected . mitoTALEN monomers positive for both eGFP mCherry were isolated as " Yellow " . " Black " represent mitoTALEN sorted population of negative for eGFP mCherry " GFP - + " population represents mitoTev - TALE sorted with low of eGFP . GFP + + were positive for GFP after mitoTev - TALEs

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MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections


of % of WT load in cybrid sorted populations after

Quantification of the % of WT load in cybrid sorted populations after transfection


Representative RFLP showing change in heteroplasmy at 1 day 15 days , after

Representative RFLP analysis showing the change in heteroplasmy at 1 day and 15 days, after sorting


Quantification of the heteroplasmic shift over time


Determination of of mtDNA ( ND1 / ACTB ) by qPCR of mutant cybrids transfected with MERRF mitoTev -

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Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE


mtDNA in wild - type sorted transfected with MERRF mitoTev - TALE . Data is expressed as of Untransfected ( % UNT )

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mtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT)


RFLP analyses of high mutant cybrids transfected with MERRF mitoTev - TALE . sorted were analyzed at different times of growth by PCR / RFLP as previously

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RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev-TALE. The sorted cells were analyzed at different times of growth by PCR/RFLP as previously described


of WT loads of 6 - 8 independent experiments / sortings , analyzed at different time -

Quantification of WT loads of 6-8 independent experiments/sortings, analyzed at different time-points


Total mtDNA determined by qPCR in 6 - 7 separate sortings /

Total mtDNA levels determined by qPCR in 6-7 separate sortings/experiments


Flow cytometry diagram of gated sorted . GFP - + represent with low of , GFP + + showed intermediate of while GFP + + + had highest

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Flow cytometry diagram of gated sorted cells. The GFP-+ represent the cells with low levels of fluorescence, GFP++ showed intermediate levels of fluorescence while GFP+++ had the highest levels


of mtDNA by PrimeTime qPCR probes . mtDNA were analyzed in different populations of sorted . Data are expressed as mean ± of 3 - 4 separate experiments mtDNA were also determined in WT cybrids as described above . Data are expressed as mean ± of 4 - 7 separate

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Quantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean±SEM of 3-4 separate experiments mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean±SEM of 4-7 separate experiments


Oxygen consumption rate ( OCR ) upon sequential injection of oligomycin ( Oligo ) , FCCP rotenone ( Rot ) + antimycin ( AA ) , in untransfected , sorted " GFP - + " " GFP + + " . error bars represent mean ± of 5 - 7 separate

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Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experiments


of 4 - 6 independent experiments comparing basal respiration , maximal respiration , spare respiratory capacity ATP - linked respiration of UNT versus " GFP - + " , " GFP + + " WT . were analyzed between 25 - 28 days after

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Quantification of 4-6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration of the UNT versus "GFP-+", "GFP++" and WT cells. The cells were analyzed between 25-28 days after sorting


Coupling efficiency , measured as of ATP - linked / Basal respiration x 100 . Error bars represent mean ± of 4 - 6 separate

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Coupling efficiency, measured as the ratio of ATP-linked/Basal respiration x 100. Error bars represent the mean±SEM of 4-6 separate experiments


Representative of a mitochondrial synthesis experiment . results were normalized to gel

Representative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loading


Western - blot showing of NDUFB8 COXI in untransfected sorted . SDHA tubulin were also analyzed . panel also shows of these markers normalized by stain - free loading of 6 independent sortings /

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Western-blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of 6 independent sortings/experiments


( G ) Sorted human monocytes were incubated with control medium or SARS - CoV - 2 at an MOI of 1 for 1 h , followed by washing away extracellular further culture for 1 h . were fixed stained with indicated antibodies . Otherwise , monocytes were challenged with 1 μg / ml LPS for 3 h 10 μM nigericin for 30 min , followed by fixing with indicated antibodies . Treatment with medium from uninfected Vero served as a control . Scale bar : 5 μm . Data information : results were repeated for three times with similar results .

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(G) Sorted human monocytes were incubated with control medium or SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS for 3 h and 10 μM nigericin for 30 min, followed by fixing and staining with the indicated antibodies. Treatment with medium from uninfected Vero cells served as a control. Scale bar: 5 μm. Data information: results were repeated for three times with similar results.


Supernatants from control or SARS - CoV - 2 infected CD14 + monocytes with or without LPS nigericin stimulation were collected subjected to an ELISA for IL - 1β ( H ) . Student ' s t - was used data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : , Student ' s t - was used data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results .

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Supernatants from control or SARS-CoV-2 infected CD14+ monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL-1β levels (H). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: , Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.


viability in pellets were examined using a viability through checking ATP inside ( I ) . of undergoing pyroptosis was calculated through checking released LDH in supernatants ( J ) . Student ' s t - was used data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : Student ' s t - was used data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results .

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Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (J). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.


( B , C ) THP - 1 stably expressing indicated SARS - CoV - 2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were collected for an ELISA to determine supernatant IL - 1β ( B ) pellets were subjected to a viability to verify of viable through checking ATP ( C ) . For B - C , Student ' s t - was used data were shown as means ± SD of three technical replicates . NS , non - significant . Data information : control vector used here was empty vector corresponded to nucleocapsid - containing vector . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times .

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(B, C) THP-1 cells stably expressing the indicated SARS-CoV-2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine supernatant IL-1β levels (B) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels (C). For B-C, Student's t-test was used and data were shown as means±SD of three technical replicates. NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.


( D - F ) THP - 1 stably expressing nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants pellets were collected immunoblotted with antibodies against indicated ( D ) . of viable was determined through checking ATP in pellets ( E ) . of undergoing pyroptosis was calculated through checking released LDH in supernatants ( F ) . For E , F , Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Data information : control vector used here was empty vector corresponded to nucleocapsid - containing vector . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times .

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(D-F) THP-1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins (D). The percentage of viable cells was determined through checking cellular ATP levels in cell pellets (E). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.


( D ) GSDMD + / + GSDMD - / - THP - 1 were transfected with plasmids encoding nucleocapsid for 24 h , followed by fixation , permeabilization stain with antibodies against nucleocapsid GSDMD . nucleus was counterstained with DAPI . Scale bar : 5 μm .

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(D) GSDMD+/+ and GSDMD-/- THP-1 cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain with antibodies against nucleocapsid and GSDMD. The nucleus was counterstained with DAPI. Scale bar: 5 μm.


( E , F ) THP - 1 ( E ) or human CD14 + monocytes ( F ) were transfected with plasmids encoding FLAG - tagged nucleocapsid for 36 h , followed by pretreatment of 1 μg / ml LPS for 3 h 10 μM nigericin stimulation for 30 min . were lysed immunoprecipitated with antibody against FLAG or isotype IgG . Input precipitates were immunoblotted as indicated .

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(E, F) THP-1 (E) or human CD14+ monocytes (F) were transfected with plasmids encoding FLAG-tagged nucleocapsid for 36 h, followed by pretreatment of 1 μg/ml LPS for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.


( I ) GST - GSDMD control GST were mixed with HEK293T lysate containing FLAG - tagged full - nucleocapsid or truncated nucleocapsid lacking 70 - 160 290 - 360 , followed by a GST - pulldown . Input precipitates were immunoblotted as indicated .

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(I) GST-GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG-tagged full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted as indicated.


( A , B ) THP - 1 stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . were either immunoblotted with antibodies against indicated ( A ) or stained with propidium iodide ( PI ) ( B , left ) . Scale bar : 20 μm . of PI positive was calculated ( B , right ) . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to nucleocapsid - containing vector . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times .

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(A, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.


( H , I ) THP - 1 stably expressing full - nucleocapsid or mutant lacking 70 - 160 290 - 360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . were either immunoblotted with antibodies against indicated ( H ) . were also stained with PI of PI positive was calculated ( I ) . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to nucleocapsid - containing vector . Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times .

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(H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.


( A ) GST or GST - GSDMD , p20 ( C285A ) - His , p10 - His increasing amount of His - nucleocapsid were incubated for a GST - pulldown , followed by immunoblotting with antibodies against indicated tags . Data information : Experiments were repeated for three times with similar results .

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(A) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results.


( G , H ) Wild - type GSDMD GSDMDsusti . ( G ) or mCherry - GSDMD - C ( H ) were purified from HEK293T incubated with recombinant p20 / p10 His - tagged nucleocapsid in a cleavage buffer , followed by immunoblotting with antibodies against indicated tags or antibody against GSDMD C - termimus . Data information : Experiments were repeated for three times with similar results .

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(G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus. Data information: Experiments were repeated for three times with similar results.


GSDMD - / - THP - 1 rescued with wild - type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . were immunoblotted with antibodies against indicated ( A )

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GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (A)


( E , F ) GSDMD - / - THP - 1 rescued with wild - type or linker substituted GSDMD were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular further culture for 3 h . Otherwise , were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were subjected to an ELISA to determine IL - 1β ( E ) . Otherwise , viability in pellets was examined using a viability through checking ATP inside ( F ) . For E , F , Student ' s t - was used data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results .

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(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.


( G ) Sorted CD14 + monocytes from donors were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular further culture for 3 h . were immunoblotted with antibodies against indicated . CD14 + monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h served as a positive control . Data information : experiments were repeated for three time with similar results .

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(G) Sorted CD14+ monocytes from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control. Data information: experiments were repeated for three time with similar results.


( B ) Lineage tracing of anagen matrix from β - actin - CreER : Confetti , 7 days after induction with either a high dose of tamoxifen ( TM ) ( top ) or low dose ( bottom ) for single clonal lineages .

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(B) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages.


( C ) of skin sections at various stages of hair cycle , immunostained for Gata6 ( red ) , CD34 or K14 ( green ) ( blue ) .

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(C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue).


( D ) ( D ' ) for Gata6 inducible knockout ( iKO ) wild type ( WT ) mice over time , showing loss of Gata6 ( red ) from hair follicle . Scale bars : 30 m .

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(D) images (D') for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.


( B ) Representative of hematoxylin of skin sections from telogen induced mice sacrificed at 2 , 4 , 10 days ( d ) post TM induction . ( C ) of hematoxylin of skin section from anagen induced mice . shown in Figure EV2D . Scale bars : 30 m .

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(B) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.


( B - G ) Gata6 WT iKO skin after 3 days after TM induction 12 hours after BrdU labeling or 5 days after TM induction 2 days after BrdU labeling stained with markers as indicated in corresponding color .

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(B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.


( A ) Hair follicles exhibit immunostaining signal for damage marker H2A . X ( red ) apoptosis marker Caspase3 ( green ) as early as one day after Gata6 iKO induction . Top panel shows a section of UV irradiated epidermis as a positive control for damage . Note that Caspase 3 is not expressed in H2A . X of epidermis but is found in some iKO hair follicle matrix . Right panels show single color as indicated at top ( B ) ( average SD ) of of apoptotic per matrix as determined by counting of Caspase3 + ( n = 50 follicles from 3 mice per group ) ( C ) ( average SD ) of of damaged per matrix as determined by counting of H2A . X + Caspase3 - ( n = 50 follicles from 3 mice per group ) .

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(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group).


( D ) Regional distribution of apoptosis was analyzed by dividing matrix into four compartments as shown in legend ( left ) counting fraction of Caspase3 + per each matrix compartments ( right ; n = 50 follicles from 3 mice per group ) .

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(D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group).


( E ) Proliferation in matrix compartments shown in ( D ) was assessed by BrdU labeling for 1 hour , followed by , ( F ) ( interior labeled with solid bars , exterior labeled with striped bars ) . Unpaired t - : * P - value < 0 . 05 . n = 20 follicles from 2 mice per group .

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(E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.


( A ) Colony formation of WT TM induced Gata6 iKO keratinocytes stained with hematoxylin . keratinocytes were isolated from induced iKO newborn mice , plated on feeder , stained with hematoxylin at day 7 after plating ( top ) . Phase contrast of are shown below . Scale bars : 30 m . ( B ) of colony formation from ( A ) , average SD from n = 3 mice in each genotype .

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(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype.


( C ) Western blot of WT iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction , blotted with actin Gata6 antibodies , show a decrease in Gata6 after 6 hours .

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(C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours.


( D ) Phase contrast of WT Gata6 iKO keratinocytes 3 days after induction with TM ( n > 3 per genotype ) .

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(D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype).


( E - H ) of damage in cultured keratinocytes on for damage marker H2A . X ( E G ) comet ( F H ) . Arrowheads in ( F ) indicate damaged nuclei with comet shape . of H2A . X ( G ) represents average SD from at least 300 per condition from 3 experiments using different lines . * Unpaired t - P - values : 6h = 0 . 01 24h = 0 . 004 . ( H ) of comet represents average SD ( n = 3 ) .

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(E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3).


( I - J ) FACS cycle profiles show that Gata6 iKO keratinocytes show decreased proliferation G2 - M arrest .

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(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest.


( I - J ) FACS cycle profiles show that Gata6 iKO keratinocytes show decreased proliferation G2 - M arrest . ( J ) of FACS cycle represent average SD ( n = 3 per genotype ) . * Unpaired t - P - values : G1 = 0 . 03 , S = 5x10 - 4 , G2 - M = 7x10 - 4 .

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(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4.


( K ) of frequency of H2A . X + in keratinocytes within S phase ( EdU + ) or another cycle stage ( EdU - ) following a 20 minute EdU labeling ( average SD , n = 5 ) . * Unpaired t - P - values WT = 0 . 1 , iKO = 0 . 05 . Mann - Whitney U P - values : WT = 0 . 09 , iKO = 0 . 03 .

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(K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03.


( L - M ) Cultured keratinocytes were made quiescent by gradual serum depletion . ( L ) of EdU + in proliferative serum depleted quiescent ( average SD , n = 4 ) . ( M ) of H2A . X + keratinocytes during proliferative quiescent conditions ( average SD , n = 4 ) . * Unpaired t - P - value = 0 . 04 . Mann - Whitney U P - value = 0 . 06 .

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(L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.


( A ) of differentially expressed genes ( adjusted P - value < 0 . 05 fold change > 1 . 5 ) from - seq ( n = 3 different WT iKO lines ) .

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(A) Heatmap of differentially expressed genes (adjusted P-value < 0.05 and fold change > 1.5) from RNA-seq (n=3 different WT and iKO cell lines).


( B ) qRT - PCR validation of candidate genes selected from - seq using from cultured ( average SD , n = 4 ) . * Unpaired t - P - values : Edaradd = 0 . 03 , Apcdd1 = 0 . 005 , Gls2 = 7x10 - 5 , Ppargc1b = 3x10 - 4 , Rab17 = 2x10 - 5 , Foxn1 = 0 . 03 . Mann - Whitney U P - values : Edaradd = 0 . 06 , Apcdd1 = 0 . 03 , Gls2 = 0 . 03 , Ppargc1b = 0 . 03 , Rab17 = 0 . 03 , Foxn1 = 0 . 03 . ( C ) Table summarizing selection of candidate genes from ( B ) . Genes were selected on known function or skin phenotype related to Gata6 iKO phenotype .

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(B) qRT-PCR validation of candidate genes selected from RNA-seq analysis using RNA from cultured cells (average SD, n=4). *Unpaired t-test P-values: Edaradd=0.03, Apcdd1=0.005, Gls2=7x10-5, Ppargc1b=3x10-4, Rab17=2x10-5, Foxn1=0.03. Mann-Whitney U test P-values: Edaradd=0.06, Apcdd1=0.03, Gls2=0.03, Ppargc1b=0.03, Rab17=0.03, Foxn1=0.03. (C) Table summarizing selection of candidate genes from (B). Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype.


( D ) of ROS by DHE . LY83583 treated served as positive control with elevated ROS . ( E ) of fold change in ROS at 6 24 hours after TM compared to WT , on of DHE ( average SD , n = 2 ) .

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(D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2).


( F ) of transcription factor binding site overrepresentation ( oPOSSUM ) in differentially expressed genes from - seq . Points represent transcription factors plotted as Z . score of frequency of transcription factor binding site occurrences in promoters of downregulated genes compared to background rate of occurrences in gene promoters Fisher P - value of proportion of downregulated gene promoters containing each transcription factor binding site compared to frequency in gene promoters .

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(F) Analysis of transcription factor binding site overrepresentation (oPOSSUM) in differentially expressed genes from RNA-seq. Points represent transcription factors plotted as Z.score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P-value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters.


( H ) qRT - PCR of selected genes found to be overlapping between Gata6 iKO Eda transgenic analyzed in Gata6 WT iKO keratinocytes ( average SD , n = 4 ) . * Unpaired t - P - values : Mcm3 = 0 . 001 , Mcm6 = 0 . 02 , Mcm10 = 0 . 04 , Exo1 = 0 . 02 , Pold1 = 0 . 02 . Mann - Whitney U P - values : Mcm3 = 0 . 03 , Mcm6 = 0 . 03 , Mcm10 = 0 . 03 , Exo1 = 0 . 03 , Pold1 = 0 . 06 .

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(H) qRT-PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes (average SD, n=4). *Unpaired t-test P-values: Mcm3=0.001, Mcm6=0.02, Mcm10=0.04, Exo1=0.02, Pold1=0.02. Mann-Whitney U test P-values: Mcm3=0.03, Mcm6=0.03, Mcm10=0.03, Exo1=0.03, Pold1=0.06.


( B ) qRT - PCR confirmation of change in Edaradd mRNA expression in - vivo from mouse whole skin ( average SD , 1d n = 3 , 2d n = 4 ) . * Unpaired t - P - values : 1d = 0 . 02 , 2d = 0 . 05 . Mann - Whitney U P - values : 1d = 0 . 06 , 2d = 0 . 03 .

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(B) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03.


( C ) ChIP - qPCR for Gata6 at three conserved binding sites within Edaradd promoter . GAPDH served as a negative control . Binding site 1 , 2 , 3 are located 50 bp , 1 kb , 1 . 5 kb upstream of transcription start site , respectively ( average SD , n = 4 ) . * Unpaired t - P - values GAPDH = 0 . 4 , 1 = 0 . 002 , 2 = 0 . 8 , 3 = 0 . 2 . Mann - Whitney U P - values : GAPDH = 0 . 3 , 1 = 0 . 06 , 2 = 0 . 9 , 3 = 0 . 2 .

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(C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2.


( D ) Western blot from whole skin lysate . Note decrease of Edaradd activated pNF - B in iKO .

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(D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO.


( E - F ) for Edaradd show decrease in Edaradd in matrix of Gata6 iKO . * Unpaired t - P - value = 3 . 5x10 - 14 . n = 100 follicles from 3 mice per genotype .

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(E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype.


( G - H ) for pNF - B show decrease of active NF - B in Gata6 iKO matrix . * Unpaired t - P - value = 2 . 2x10 - 16 . n = 50 follicles from 3 mice .

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(G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice.


( I ) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd . plates of keratinocytes are stained with Rhodanile Blue ( n = 3 lines each ) .

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(I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each).


( J ) of H2A . X in stably transfected with pMock , pGata6 , or pEdaradd plasmids , followed by induction with TM for 6 hours ( average SD , n = 4 with > 500 counted per condition ) . * Unpaired t - P - values = 2x10 - 4 ( iKO pGata6 vs pMock ) , 2x10 - 4 ( iKO pEdaradd vs pMock ) .

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(J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock).


( K ) qRT - PCR of Mcm10 expression in pMock , pGata6 , pEdaradd WT iKO expressing ( average SD , n = 4 ) . * Unpaired t - P - values : 8x10 - 4 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) . Mann - Whitney U P - values : 0 . 03 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) .

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(K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).