( 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 .

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

(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 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 ) .

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

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 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 ) .

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

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).


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 .

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

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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ,

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

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 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

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

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 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

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

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


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


Representative RFLP analysis showing the change in heteroplasmy at 1 day and 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 the levels of mtDNA ( ND1 / ACTB ) by qPCR of mutant cybrids transfected with MERRF mitoTev -

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

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


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

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

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 the MERRF mitoTev - TALE . The sorted cells were analyzed at different times of growth by PCR / RFLP as previously

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

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


Quantification 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 levels 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 cells . The GFP - + represent the cells with low levels of fluorescence , GFP + + showed intermediate levels of fluorescence while GFP + + + had the highest

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

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


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

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

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 and rotenone ( Rot ) + antimycin ( AA ) , in untransfected , sorted " GFP - + " and " GFP + + " . The error bars represent the mean ± SEM of 5 - 7 separate

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

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


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

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

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 the ratio of ATP - linked / Basal respiration x 100 . Error bars represent the mean ± SEM of 4 - 6 separate

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

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

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


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 /

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

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 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 .

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

(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 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 .

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

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.


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 .

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

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 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 .

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

(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 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 .

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

(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 + / + 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 .

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

(D) 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 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 .

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

(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 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 .

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

(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 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 .

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

(A, B) 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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(G, H) 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 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 )

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

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 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 .

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

(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 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 .

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

(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 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 .

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

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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 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 .

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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 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 .

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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 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 ) .

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

(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 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 ) .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O 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) 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 images of WT and 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 ) 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 ) .

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

(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 cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and 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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 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 .

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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 ) 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and activated pNF - B in the 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 ) 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 .

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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 ) 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 .

(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 . Cell plates of keratinocytes are stained with Rhodanile Blue ( n = 3 cell 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 ) 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 ) .

(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 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 ) .

(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).


E . RT - qPCR of Axin2 in bEnd . 3 cells treated with NDP ( 200 ng / ml ) , isotype control or F4L5 . 13 ( 1200 ng / ml ) and transfected with control , Tspan12 or Fzd4 siRNAs . Data are presented as mean ± SD , n = 3 technical replicates . Data is representative of two independent experiments . F . Time course of phosphorylated Disheveled - 3 ( p - DVL3 ) and ßcatenin protein levels in bEnd . 3 cells treated with 30nM of F4L5 . 13 or NDP . Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß - Tubulin measured by densitometry of independent experiments . Data are presented as mean ± SEM , n = 4 - 6 ( * p < 0 . 05 as compared with NT ) . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( * p < 0 . 05 as compared with NT ) .

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n=3 technical replicates. Data is representative of two independent experiments. F. Time course of phosphorylated Disheveled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n=4-6 (*p<0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT).


A . Volcano plot of gene expression changes in bEnd . 3 cells following 24h of treatment with 30nM F4L5 . 13 . Genes with significant changes in expression level are highlighted in blue and genes co - regulated by treatment with 30nM NDP for 24h are highlighted in red . Statistical analysis was performed using the DESeq2 R package and adjusted p - value ( Wald test ) threshold less than 0 . 1 was used . Labelled genes are associated with enriched GO biological processes indicated below .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot of gene expression changes in bEnd.3 cells following 24h of treatment with 30nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30nM NDP for 24h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted p-value (Wald test) threshold less than 0.1 was used. Labelled genes are associated with enriched GO biological processes indicated below.


B . Immunofluorescence of ZO - 1 , CLDN3 and CLDN5 localization at bEnd . 3 cell junctions . bEnd . 3 cells were treated or not with 30 nM F4L5 . 13 or 30 nM Norrin ( NDP ) with or without 100 ng / ml VEGF for 1h . ZO - 1 is shown in green , CLDN3 / 5 in red and DAPI in blue . Scale bars : 15 µm . C . Quantification of ZO - 1 , CLDN3 and CLDN5 fluorescence intensity . Each column represents 40 measurements of fluorescence intensity per condition ( y - axis ) . Data are presented as mean ± SEM . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( * p < 0 . 05 as compared to VEGF treatment ) . D

O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of ZO-1, CLDN3 and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1h. ZO-1 is shown in green, CLDN3/5 in red and DAPI in blue. Scale bars: 15 µm. C. Quantification of ZO-1, CLDN3 and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment). D


C . IB4 - stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5 . 13 treatment . Boxed areas are shown enlarged in the panels on the right . Scale bars : 500 µm . D . Quantification of the vascularized area in the outer plexiform layer . 10 mg / kg F4L5 . 13 or isotype control antibody was injected every 48h from P5 to P20 . Data are presented as mean ± SD , n = 5 retinas ( 2 - 4 fields of view per retina ) from 4 - 5 mice per group . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test . E

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 O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IB4-stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5.13 treatment. Boxed areas are shown enlarged in the panels on the right. Scale bars: 500 µm. D. Quantification of the vascularized area in the outer plexiform layer. 10 mg/kg F4L5.13 or isotype control antibody was injected every 48h from P5 to P20. Data are presented as mean± SD, n=5 retinas (2-4 fields of view per retina) from 4-5 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test. E


E . F4L5 . 13 rescues barrier function defects in Tspan12 - / - mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP . IB4 - Alexa647 was used to stain ECs . Scale bars : 100 µm .

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E. F4L5.13 rescues barrier function defects in Tspan12-/- mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP. IB4-Alexa647 was used to stain ECs. Scale bars: 100 µm.


D . Flat - mounted retinas of adult Tspan12 - / - mice injected systemically with sulfo - NHS - biotin show that F4L5 . 13 partially restored BRB function . PECAM was used to stain endothelial cells . Images are representative of 4 retinas per group . Scale bar : 500 µm .

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D. Flat-mounted retinas of adult Tspan12-/- mice injected systemically with sulfo-NHS-biotin show that F4L5.13 partially restored BRB function. PECAM was used to stain endothelial cells. Images are representative of 4 retinas per group. Scale bar: 500 µm.


E . Maximum intensity projection of adult Tspan12 - / - retinas after mice were injected with F4L5 . 13 or isotype control . Endothelial cells were stained with IB4 - Alexa 647 . Images are representative of 4 retinas per group . Scale bar : 100 µm .

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E. Maximum intensity projection of adult Tspan12-/- retinas after mice were injected with F4L5.13 or isotype control. Endothelial cells were stained with IB4-Alexa 647. Images are representative of 4 retinas per group. Scale bar: 100 µm.


A . Schematic diagram of the OIR model ( top ) and representative images of P17 OIR retinas are shown ( bottom ) . Neonatal mice were exposed to 75 % oxygen from P7 to P12 to induce vessel loss . Then mice received intravitreal injections of PBS vehicle , mouse - specific anti - VEGF ( as a positive control ) delivered at 0 . 1 μg / μl , or F4L5 . 13v2 ( a second - generation F4L5 . 13 modality , in which the N - terminal FZD4 - specific diabody was replaced with a FZD4 - specific Fab ) at 50nM or 500nM target vitreous concentrations . Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17 . At P17 , retinas from injected mice were collected , dissected as flat ‐ mounts and stained with Isolectin B4 . Scale bar = 500 µm . B . Quantification of the ratio of neovascular and avascular area on total area of vascularization . Data are presented as mean ± SEM , n = 11 - 13 retinas per group . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( indicated p - values are in comparison to Vehicle ) . E

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 μg/μl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50nM or 500nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat‐mounts and stained with Isolectin B4. Scale bar = 500 µm. B. Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n=11-13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (indicated p-values are in comparison to Vehicle). E


A Measurement of urinary albumin / creatinine by ELISA ( albumin - to - creatinine ratio : day 14 : Phb2fl / fl 0 . 09 ± 0 . 03 mg / mg , n = 4 , versus Phb2pko 0 . 37 ± 0 . 21 mg / mg , n = 4 , P = 0 . 2350 ; day 21 : Phb2fl / fl 0 . 65 ± 0 . 16 mg / mg , n = 4 , versus Phb2pko 193 . 10 ± 26 . 81 mg / mg , n = 4 , * * * P = 0 . 003 ; day 28 : Phb2fl / fl 0 . 57 ± 0 . 07 mg / mg , n = 4 , versus Phb2pko 470 . 40 ± 131 . 30 mg / mg , n = 4 , * P = 0 . 0117 ) .

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A Measurement of urinary albumin/creatinine by ELISA (albumin-to-creatinine ratio: day 14: Phb2fl/fl 0.09 ± 0.03 mg/mg, n = 4, versus Phb2pko 0.37 ± 0.21 mg/mg, n = 4, P = 0.2350; day 21: Phb2fl/fl 0.65 ± 0.16 mg/mg, n = 4, versus Phb2pko 193.10 ± 26.81 mg/mg, n = 4, ***P = 0.003; day 28: Phb2fl/fl 0.57 ± 0.07 mg/mg, n = 4, versus Phb2pko 470.40 ± 131.30 mg/mg, n = 4, *P = 0.0117).


B Appearance of Phb2fl / fl and Phb2pko mice at day 28 .

B Appearance of Phb2fl/fl and Phb2pko mice at day 28.


C Analysis of body weight from postnatal day 14 until day 32 ( P - values for Phb2fl / fl or Phb2het versus Phb2pko : * P = 0 . 0372 at day 27 , * P = 0 . 0311 at day 28 , * P = 0 . 0157 at day 29 , * * P = 0 . 008 at day 30 , * * P = 0 . 0013 at day 31 , * * P = 0 . 0035 at day 32 , n = 3 for Phb2fl / fl and n = 4 for Phb2het and Phb2pko ) .

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C Analysis of body weight from postnatal day 14 until day 32 (P-values for Phb2fl/fl or Phb2het versus Phb2pko: *P = 0.0372 at day 27, *P = 0.0311 at day 28, *P = 0.0157 at day 29, **P = 0.008 at day 30, **P = 0.0013 at day 31, **P = 0.0035 at day 32, n = 3 for Phb2fl/fl and n = 4 for Phb2het and Phb2pko).


D Measurement of serum creatinine levels of mice in their fifth week of life ( Phb2fl / fl / het 14 . 74 ± 3 . 75 μmol / l versus Phb2pko 74 . 73 ± 2 . 20 μmol / l , n = 3 for both groups ; * * * P = 0 . 0002 ) .

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D Measurement of serum creatinine levels of mice in their fifth week of life (Phb2fl/fl/het 14.74 ± 3.75 μmol/l versus Phb2pko 74.73 ± 2.20 μmol/l, n = 3 for both groups; ***P = 0.0002).


E Measurement of serum urea levels of mice in their fifth week of life ( Phb2fl / fl / het 44 . 00 ± 7 . 02 mg / dl versus Phb2pko 493 . 70 ± 45 . 34 mg / dl , n = 3 for both groups ; * * * P = 0 . 006 ) .

E Measurement of serum urea levels of mice in their fifth week of life (Phb2fl/fl/het 44.00 ± 7.02 mg/dl versus Phb2pko 493.70 ± 45.34 mg/dl, n = 3 for both groups; ***P = 0.006).


F Kaplan - Meier survival curve ( n = 5 for Phb2fl / fl / het and n = 6 for Phb2pko ) .

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F Kaplan-Meier survival curve (n = 5 for Phb2fl/fl/het and n = 6 for Phb2pko).


Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections ( scale bar : 20 μm ) . C Analysis of podocyte foot processes in electron micrographs ( scale bar : 0 . 7 μm ) . FP , podocyte foot processes ; GBM , glomerular basement membrane ; EC , endothelial cells . D Analysis of mitochondrial morphology in electron micrographs ( arrows ; scale bar : 0 . 3 μm ) . E High - power view of mitochondria in electron micrographs ( scale bar : 0 . 1 μm ) .

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Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections (scale bar: 20 μm).C Analysis of podocyte foot processes in electron micrographs (scale bar: 0.7 μm). FP, podocyte foot processes; GBM, glomerular basement membrane; EC, endothelial cells.D Analysis of mitochondrial morphology in electron micrographs (arrows; scale bar: 0.3 μm).E High-power view of mitochondria in electron micrographs (scale bar: 0.1 μm).


A PAS staining of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) .

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A PAS staining of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).


C Coomassie stain of urinary samples of tPod - Phb2pko and control mice .

C Coomassie stain of urinary samples of tPod-Phb2pko and control mice.


A Kaplan - Meier survival curve showed that an additional knockout of the insulin receptor ( Insr ) prolonged survival of Phb2pko mice ( n = 7 for Phb2pko / Insrpko , n = 4 for Phb2pko / Insrhet , and n = 6 for Phb2pko ) .

A Kaplan-Meier survival curve showed that an additional knockout of the insulin receptor (Insr) prolonged survival of Phb2pko mice (n = 7 for Phb2pko/Insrpko, n = 4 for Phb2pko/Insrhet, and n = 6 for Phb2pko).


B Kaplan - Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency ( n = 6 for Phb2pko / Igf1rpko , n = 4 for Phb2pko / Igf1rhet , and n = 6 for Phb2pko ) .

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B Kaplan-Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency (n = 6 for Phb2pko/Igf1rpko, n = 4 for Phb2pko/Igf1rhet, and n = 6 for Phb2pko).


C Statistical analysis comparing all genotypes with Phb2pko mice .

C Statistical analysis comparing all genotypes with Phb2pko mice.


D Kaplan - Meier survival curve revealed prolonged survival of Phb2pko / Insrpko / Igf1rpko and Phb2pko / Insrpko / Igf1rhet mice ( n = 19 for Phb2pko / Insrpko / Igf1rpko , n = 9 for Phb2pko / Insrhet / Igf1rpko , n = 11 for Phb2pko / Insrpko / Igf1rhet , n = 9 for Phb2pko / Insrhet / Igf1rhet , and n = 6 for Phb2pko ) . E Statistical analysis comparing all genotypes with Phb2pko mice .

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D Kaplan-Meier survival curve revealed prolonged survival of Phb2pko/Insrpko/Igf1rpko and Phb2pko/Insrpko/Igf1rhet mice (n = 19 for Phb2pko/Insrpko/Igf1rpko, n = 9 for Phb2pko/Insrhet/Igf1rpko, n = 11 for Phb2pko/Insrpko/Igf1rhet, n = 9 for Phb2pko/Insrhet/Igf1rhet, and n = 6 for Phb2pko).E Statistical analysis comparing all genotypes with Phb2pko mice.


A Serum creatinine and urea levels in the fifth week of life ( left graph : Phb2pko 74 . 73 ± 2 . 20 μmol / l versus Phb2pko / Insrpko / Igf1rpko 37 . 36 ± 8 . 83 μmol / l , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0169 ; right graph : Phb2pko 493 . 70 ± 45 . 34 mg / dl versus Phb2pko / Insrpko / Igf1rpko 272 . 80 ± 49 . 29 mg / dl , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0247 ) .

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum creatinine and urea levels in the fifth week of life (left graph: Phb2pko 74.73 ± 2.20 μmol/l versus Phb2pko/Insrpko/Igf1rpko 37.36 ± 8.83 μmol/l, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0169; right graph: Phb2pko 493.70 ± 45.34 mg/dl versus Phb2pko/Insrpko/Igf1rpko 272.80 ± 49.29 mg/dl, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0247).


B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl / fl , Phb2het , Phb2pko and Phb2pko / Insrpko / Igf1rpko mice compared to the floxed Phb2 gene ( left graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 83 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) and compared to a reference gene ( right graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 58 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) .

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B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl/fl, Phb2het, Phb2pko and Phb2pko/Insrpko/Igf1rpko mice compared to the floxed Phb2 gene (left graph; quantified by qPCR: n = 3 for all groups, P = 0.83 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko) and compared to a reference gene (right graph; quantified by qPCR: n = 3 for all groups, P = 0.58 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko).


C Analysis of mitochondrial morphology in electron micrographs ( left image : scale bar , 1 μm ; right image : scale bar , 0 . 1 μm ) .

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C Analysis of mitochondrial morphology in electron micrographs (left image: scale bar, 1 μm; right image: scale bar, 0.1 μm).


A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2 - deficient podocytes ( scale bar : 10 μm ) . B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in ( A ) ( mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed ) . C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . D Morphometric image analysis showed a decrease in branch numbers in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . E Morphometric image analysis detected a decrease in mitochondrial size in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2-deficient podocytes (scale bar: 10 μm).B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in (A) (mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed).C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).D Morphometric image analysis showed a decrease in branch numbers in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).E Morphometric image analysis detected a decrease in mitochondrial size in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).


F Measurements of mitochondrial respiration with complex II substrates ( succinate and glycerol - 3 - phosphate ) did not show significant differences between Phb2 - deficient and control podocytes ( n = 6 ) .

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F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).


G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2 - deficient podocytes compared to control podocytes ( n = 3 ) .

G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3).


H FACS analysis of MitoTracker - and MitoSOX - stained Phb2 - deficient podocytes and control podocytes . I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2 - deficient podocytes compared to control podocytes ( n = 3 , P = 0 . 7782 for MitoTracker , P = 0 . 1088 for MitoSOX ) .

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H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).


A Phb2 - deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes , which could be blocked by treatment with rapamycin ( n = 5 , bars represent mean ± SEM , Student ' s t - test , * * P = 0 . 0036 ) . pS6RP , phosphorylated S6 ribosomal protein ; S6RP , S6 ribosomal protein .

O 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

A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.


B Immunohistochemistry for phosphorylated S6 ribosomal protein ( pS6RP ) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl / fl mice ( arrows point to cells showing a signal for pS6RP ; scale bar : 20 μm ) .

O O O O O O O O O O O O O O O O O O O O O O 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 Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm).


C Kaplan - Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle - treated Phb2pko mice ( n = 11 for Phb2fl / fl + rapamycin , n = 15 for Phb2pko + vehicle , n = 16 for Phb2pko + rapamycin ) . D Statistical analysis comparing rapamycin - treated Phb2pko mice with vehicle - treated Phb2pko mice .

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

C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.


( C ) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells ( upper panel ) . The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells (upper panel). The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel (n = 3 independent experiments).


( C ) Survival curves of mice transplanted with CD4 + T cells expressing the indicated proteins . The number of animals used is indicated in the graph . Note : the mouse transplanted with EGFP - transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival curves of mice transplanted with CD4+ T cells expressing the indicated proteins. The number of animals used is indicated in the graph. Note: the mouse transplanted with EGFP-transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological analyses


( F ) Quantification of CD4 + versus CD8 + T cell ratio in peripheral blood from mice transplanted with CD4 + T cells expressing the indicated proteins ( bottom ) . Each point represents the measurement of an individual mouse . n as in B . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(F) Quantification of CD4+ versus CD8+ T cell ratio in peripheral blood from mice transplanted with CD4+ T cells expressing the indicated proteins (bottom). Each point represents the measurement of an individual mouse. n as in B. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( J - L ) Representative flow cytometry analysis ( J ) and quantification of the surface levels of ICOS ( K ) and CD69 ( L ) in TFH cells from mice transplanted with CD4 + T cells expressing the indicated proteins . In J , numbers indicate the relative percentage ( % ) of the cell population that has been interrogated ( boxed ) . In K and L , each point represents the values obtained with a single experimental mouse . n as in H . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(J-L) Representative flow cytometry analysis (J) and quantification of the surface levels of ICOS (K) and CD69 (L) in TFH cells from mice transplanted with CD4+ T cells expressing the indicated proteins. In J, numbers indicate the relative percentage (%) of the cell population that has been interrogated (boxed). In K and L, each point represents the values obtained with a single experimental mouse. n as in H. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( M ) Abundance of indicated transcripts ( bottom ) in splenic cells from control ( EGFP + ) , tumor - bearing mice ( expressing EGFP and Vav1 ∆ C ) and mice transplanted with cells transduced with the catalytically dead version of Vav1 ∆ C ( expressing EGFP and Vav1 ∆ C + E201A ) . Values are given relative to the expression of each transcript in samples obtained from EGFP controls ( which was given an arbitrary value of 1 ) . n = 4 animals per class analyzed . a . u . , arbitrary unit . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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

(M) Abundance of indicated transcripts (bottom) in splenic cells from control (EGFP+), tumor-bearing mice (expressing EGFP and Vav1∆C) and mice transplanted with cells transduced with the catalytically dead version of Vav1∆C (expressing EGFP and Vav1∆C+E201A). Values are given relative to the expression of each transcript in samples obtained from EGFP controls (which was given an arbitrary value of 1). n = 4 animals per class analyzed. a.u., arbitrary unit. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( C ) Representative immunoblot showing the abundance of the ectopic Vav1 proteins , EGFP and the endogenous tubulin α ( loading control ) in CD4 + T - cells transduced with retrovirus particles used in these experiments ( top ) .

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

(C) Representative immunoblot showing the abundance of the ectopic Vav1 proteins, EGFP and the endogenous tubulin α (loading control) in CD4+ T-cells transduced with retrovirus particles used in these experiments (top).


( E , F ) Representative FACS plots ( E ) and quantification ( F ) of the proliferation of EGFP + CD4 + T cells expressing the indicated proteins using the Cell Trace Violet method . In E , the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4 + T cells before stimulation and retroviral transduction . In F , each point represents the values obtained with a single experimental mouse . n = 6 per each experimental condition , except in the case of Vav1 ∆ N ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

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(E, F) Representative FACS plots (E) and quantification (F) of the proliferation of EGFP+ CD4+ T cells expressing the indicated proteins using the Cell Trace Violet method. In E, the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells before stimulation and retroviral transduction. In F, each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of Vav1∆N (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( G - J ) Flow cytometry determination of Tox ( G ) , ICOS ( H ) , CD25 ( I ) and ICN1 ( J ) expression in EGFP + CD4 + T cells expressing the indicated Vav1 proteins . NSt , Non stimulated cells ( CD4 + T cells before stimulation and retroviral transduction ) . In all panels , each point represents the values obtained for a single experimental condition . f . i . , mean fluorescence intensity relative to the isotype - matched control antibody . n = 6 per each experimental condition , except for the Vav1 ∆ N and NSt conditions ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Flow cytometry determination of Tox (G), ICOS (H), CD25 (I) and ICN1 (J) expression in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. NSt, Non stimulated cells (CD4+ T cells before stimulation and retroviral transduction). In all panels, each point represents the values obtained for a single experimental condition. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 6 per each experimental condition, except for the Vav1∆N and NSt conditions (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( K - N ) Example of a flow cytometry analysis ( K and M ) and final quantification of p - Akt ( L ) and p - Erk1 / 2 ( N ) levels in EGFP + CD4 + T cells expressing the indicated Vav1 proteins in primed ( blue bars ) and restimulated ( red bars ) cells . In K and M , the grey shaded histograms represent the fluorescence obtained with the isotype - matched control antibody . In L and N , NSt , nonstimulated , St , stimulated . n = 3 per each experimental condition . Data information : values are shown as means ± SEM from three independent experiments . In panels L and N , P values are given relative to nonstimulated ( blue asterisks ) and stimulated ( red asterisks ) cells expressing Vav1WT . We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t test ) .

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

(K-N) Example of a flow cytometry analysis (K and M) and final quantification of p-Akt (L) and p-Erk1/2 (N) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the grey shaded histograms represent the fluorescence obtained with the isotype-matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition. Data information: values are shown as means ± SEM from three independent experiments. In panels L and N, P values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1WT. We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student's t test).


B ) quantification of the EGFP + CD4 + T cell proliferation in the different experimental groups n = 3 independent experiments . Data information : values are shown as means ± SEM from three independent experiments . P values are given relative to nontreated ( light blue asterisks ) and treated ( dark blue asterisks ) EGFP + cells . We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Mann - Whitney U test ) .

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B) quantification of the EGFP+ CD4+ T cell proliferation in the different experimental groups n = 3 independent experiments. Data information: values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).