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

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


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

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


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

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

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


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

O O O 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

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


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

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


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

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

O O O O O O 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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 treated with NDP ( 200 ng / ml ) , isotype control or F4L5 . 13 ( 1200 ng / ml ) 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 ) ßcatenin in bEnd . 3 treated with 30nM of F4L5 . 13 or NDP . Histogram represents of DVL3 phosphorylation over total DVL3 ßcatenin over ß - Tubulin measured by densitometry of independent experiments . Data are presented as mean ± , n = 4 - 6 ( * p < 0 . 05 as compared with NT ) . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons ( * 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 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 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

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 following 24h of treatment with 30nM F4L5 . 13 . Genes with significant changes in expression level are highlighted in blue genes co - regulated by treatment with 30nM NDP for 24h are highlighted in red . Statistical was performed using DESeq2 R package adjusted p - value ( Wald ) 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 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

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 CLDN5 localization at bEnd . 3 junctions . bEnd . 3 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 DAPI in blue . Scale bars : 15 µm . C . of ZO - 1 , CLDN3 CLDN5 . Each column represents 40 measurements of per condition ( y - axis ) . Data are presented as mean ± . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons ( * 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 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

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 restored retinal vasculature absence of misdirected angiogenesis after F4L5 . 13 treatment . Boxed areas are shown enlarged in panels on right . Scale bars : 500 µm . D . of vascularized in 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 . E

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 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

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 tight junction component CLDN5 decreased expression of EC fenestration component PLVAP . IB4 - Alexa647 was used to stain ECs . Scale bars : 100 µm .

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

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 . are representative of 4 retinas per group . Scale bar : 500 µm .

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

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

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

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 OIR model ( top ) representative 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 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 stained with Isolectin B4 . Scale bar = 500 µm . B . of of neovascular avascular on total of vascularization . Data are presented as mean ± , n = 11 - 13 retinas per group . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons ( 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 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

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 : 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 ) .

O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 Phb2pko mice at day 28 .

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


C of 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 n = 4 for Phb2het 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 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 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 n = 6 for Phb2pko ) .

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

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


Phb2pko mice develop glomerulosclerosisA PAS of kidney sections ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections ( scale bar : 20 μm ) . C of podocyte foot processes in electron ( scale bar : 0 . 7 μm ) . FP , podocyte foot processes ; GBM , glomerular basement ; EC , endothelial . D of mitochondrial in electron ( arrows ; scale bar : 0 . 3 μm ) . E High - power view of mitochondria in electron ( 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 of tPod - Phb2pko control mice 2 . 5 after end of tamoxifen treatment ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections of tPod - Phb2pko control mice 2 . 5 after 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 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 insulin receptor ( Insr ) prolonged survival of Phb2pko mice ( n = 7 for Phb2pko / Insrpko , n = 4 for Phb2pko / Insrhet , n = 6 for Phb2pko ) .

O O O O O O O O O O O O O O O O O O O O O O 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 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 survival time of Phb2pko mice is not changed by an additional Igf1r deficiency ( n = 6 for Phb2pko / Igf1rpko , n = 4 for Phb2pko / Igf1rhet , n = 6 for Phb2pko ) .

O O O O O O O O O O O O O O O O O O O O O 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 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 comparing 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 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 , n = 6 for Phb2pko ) . E Statistical comparing genotypes with Phb2pko mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 urea in 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 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 n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0247 ) .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phb2pko / Insrpko / Igf1rpko mice compared to floxed Phb2 gene ( left graph ; quantified by qPCR : n = 3 for groups , P = 0 . 83 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) compared to a reference gene ( right graph ; quantified by qPCR : n = 3 for 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 of mitochondrial in electron ( 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 of mPhb2 shRNA control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2 - deficient podocytes ( scale bar : 10 μm ) . B of tubular fragmented mitochondrial mPhb2 shRNA versus control podocytes seen in ( A ) ( mitochondrial network of n = 100 of mPhb2 shRNA podocytes n = 108 of scrambled shRNA podocytes was assessed ) . C Morphometric image revealed an increased of mitochondrial versus mitochondrial in Phb2 - deficient podocytes compared to control podocytes ( n = 75 patches for scrambled shRNA , n = 58 patches for mPhb2 shRNA ) . D Morphometric image showed a decrease in branch numbers in Phb2 - deficient podocytes compared to control podocytes ( n = 75 patches for scrambled shRNA , n = 58 patches for mPhb2 shRNA ) . E Morphometric image detected a decrease in mitochondrial in Phb2 - deficient podocytes compared to control podocytes ( n = 75 patches for scrambled shRNA , n = 58 patches for mPhb2 shRNA ) .

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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 II substrates ( succinate glycerol - 3 - phosphate ) did not show significant differences between Phb2 - deficient 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 of mtDNA as a measure for mitochondrial mass showed similar of mtDNA in Phb2 - deficient podocytes compared to control podocytes ( n = 3 ) .

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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 of MitoTracker - MitoSOX - stained Phb2 - deficient podocytes control podocytes . I of mean of MitoTracker MitoSOX by FACS revealed same mitochondrial mass same ROS 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 of phosphorylated S6 ribosomal than control podocytes , which could be blocked by treatment with rapamycin ( n = 5 , bars represent mean ± , Student ' s t - , * * P = 0 . 0036 ) . pS6RP , phosphorylated S6 ribosomal ; S6RP , S6 ribosomal .

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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 ( pS6RP ) on kidney sections revealed increased of pS6RP in glomeruli of Phb2pko compared to Phb2fl / fl mice ( arrows point to showing a signal for pS6RP ; scale bar : 20 μm ) .

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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 comparing rapamycin - treated Phb2pko mice with vehicle - treated Phb2pko mice .

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

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(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 expressing indicated . number of used is indicated in graph . Note : mouse transplanted with EGFP - transduced that has died in these experiments did not show any sign of development according to anatomopathological

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

(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 ( J ) of surface of ICOS ( K ) CD69 ( L ) in TFH from mice transplanted with CD4 + T expressing indicated . In J , numbers indicate relative ( % ) of population that has been interrogated ( boxed ) . In K L , each point represents values obtained with a single experimental mouse . n as in H . Data information : data represent mean ± . Statistical values obtained using Student ' s t are given relative to control EGFP + . * , 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 from control ( EGFP + ) , - bearing mice ( expressing EGFP Vav1 ∆ C ) mice transplanted with transduced with catalytically dead version of Vav1 ∆ C ( expressing EGFP Vav1 ∆ C + E201A ) . Values are given relative to expression of each transcript in samples obtained from EGFP controls ( which was given an arbitrary value of 1 ) . n = 4 per class analyzed . a . u . , arbitrary unit . Data information : data represent mean ± . Statistical values obtained using Student ' s t are given relative to control EGFP + . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(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 abundance of ectopic Vav1 , EGFP endogenous tubulin α ( loading control ) in CD4 + T - transduced with retrovirus particles used in these experiments ( top ) .

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(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 ) ( F ) of proliferation of EGFP + CD4 + T expressing indicated using Trace Violet method . In E , grey shaded histograms represent obtained from nonstimulated CD4 + T before stimulation retroviral transduction . In F , each point represents values obtained with a single experimental mouse . n = 6 per each experimental condition , except in case of Vav1 ∆ N ( n = 3 ) . Data information : values are shown as means ± 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 ) ICN1 ( J ) expression in EGFP + CD4 + T expressing indicated Vav1 . NSt , Non stimulated ( CD4 + T before stimulation retroviral transduction ) . In panels , each point represents values obtained for a single experimental condition . f . i . , mean relative to isotype - matched control antibody . n = 6 per each experimental condition , except for Vav1 ∆ N NSt conditions ( n = 3 ) . Data information : values are shown as means ± from three independent experiments .

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

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(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 ) of EGFP + CD4 + T proliferation in different experimental groups n = 3 independent experiments . Data information : values are shown as means ± from three independent experiments . P values are given relative to nontreated ( blue asterisks ) treated ( blue asterisks ) EGFP + . We also include P values for 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 ) .

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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).


( C - E ) Flow cytometry determination of intracellular Tox ( C ) , intracellular ICN1 ( D ) surface ICOS ( E ) in EGFP + CD4 + T expressing indicated Vav1 . f . i . , mean relative to isotype - matched control antibody . n = 3 independent experiments . Data information : values are shown as means ± from three independent experiments . P values are given relative to nontreated ( blue asterisks ) treated ( blue asterisks ) EGFP + . We also include P values for 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 ) .

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(C-E) Flow cytometry determination of intracellular Tox (C), intracellular ICN1 (D) and surface ICOS (E) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. 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).


Figure 1 : Growth Charts of Affected Subjects • A Family 1 - Subject II . 1 . • B Family 1 - Subject II . 3 . • C Family 2 - Subject II . 3 . • D Family 2 - Subject II . 4 . • E Family 2 - Subject II . 5 . • F Top Row - Representative X - rays showing narrow fibulae elongated digits in Subject II . 1 of Family 1 . Bottom Row - Lower extremities foot from Subject II . 5 from Subject II . 4 , both from Family 2 .

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Figure 1: Growth Charts and Photographs of Affected Subjects•A Family 1-Subject II.1.•B Family 1-Subject II.3.•C Family 2-Subject II.3.•D Family 2-Subject II.4.•E Family 2-Subject II.5.•F Top Row -Representative X-rays showing narrow fibulae and elongated digits in Subject II.1 of Family 1. Bottom Row - Lower extremities and foot from Subject II.5 and hand from Subject II.4, both from Family 2.


• B Pedigree representative Sanger sequencing trace of mutation identified in family 1 . • C Pedigree representative Sanger sequencing trace of mutation identified in family 2 .

• B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1.•C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2.


• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T transfected with cDNA encoding PAPP - A2 , PAPP - A2 ( D643fs ) ( family 1 ) , or PAPP - A2 ( A1033V ) ( family 2 ) . Radiolabeled substrates tested include IGFBP - 3 , IGFBP - 4 , IGFBP - 5 . Cleavage was visualized by autoradiography following SDS - PAGE . Positions of intact IGFBPs ( i ) cleavage products ( c ) are indicated .

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• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS-PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated.


B Western blots following nonreducing or reducing SDS - PAGE of media from transfected , as indicated . Polyclonal antibodies raised against N - terminal portion of PAPP - A2 were used for detection .

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B Western blots following nonreducing or reducing SDS-PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection.


• C Comparison of proteolytic activity similar to experiment of panel A , except that equimolar concentrations of PAPP - A2 PAPP - A2 ( A1033V ) were used . variant from Family 1 , carrying a frameshift mutation N - terminal to proteolytic domain of PAPP - A2 , did not show any detectable expression . gels blots are representative of three independent experiments .

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•C Comparison of proteolytic activity similar to the experiment of panel A, except that equimolar concentrations of PAPP-A2 and PAPP-A2 (A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments.


• A Concentrations of endogenous insulin - like growth factor ( IGF ) - I in different fractions obtained by neutral chromatography on a Superdex 200 column . Serum samples from four siblings of family 1 were analyzed compared to a non - related prepubertal control , as siblings were prepubertal . amount of IGF - I detected in fractions corresponding to ternary ( TC ) was markedly greater in homozygous affected subjects ( II · 1 II · 3 ) compared to unaffected siblings ( II . 2 II . 4 ) prepubertal control . No IGF - I was detected in fractions corresponding to binary ( BC ) or free IGF - I in any of subjects . This is most likely due to fact that under physiological conditions 80 - 85 % of IGF - I circulates in TC after separation procedure , amount of IGF - I in BC or free was below level of detection of our IGF - I . These results clearly demonstrate that in affected amount of IGF - I circulating in TC is markedly elevated .

• A Concentrations of endogenous insulin-like growth factor (IGF)-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a non-related prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II·1 and II·3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or free IGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated.


A . Ncl Pus7 mRNA ( normalized to TBP ) at different time points after LPS stimulation in c - mycwt / wt c - myc ∆ / ∆ . Data are presented as mean ± SD ; n = 3 .

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A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3.


B . To quantify synthesis , we exposed our cultures to a brief of ethyl - uridine ( EU ) measured its incorporation into by microscopy . scatter plots show as detected in 2D plane ( x axis , in pixels ) versus EU signal ( y axis , as arbitrary units , AU ) for each single identified by DAPI in c - mycwt / wt c - myc ∆ / ∆ populations at indicated time points after LPS treatment . One representative experiment out of 5 is shown .

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B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown.


C . Variation of each expressed mRNA at 4h of LPS treatment ( log2FC , relative to time 0 ) for c - mycwt / wt ( x - axis ) c - myc ∆ / ∆ ( y - axis ) , as measured by - seq . Regulatory groups 1 - 6 Grey Zone ( GZ ) are as defined in text .

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C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text.


D . Numbers of genes classified in different regulatory categories at indicated time points after LPS stimulation .

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D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation.


E . Variations ( log2FC ) in mature precursor RNAs ( as measured by - seq ) in modeled rates ( synthesis , processing degradation ) for Myc dependent independent genes . grey scale represents starting level for each parameter in unstimulated .

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E. Variations (log2FC) in mature and precursor RNAs (as measured by RNA-seq) and in the modeled rates (RNA synthesis, processing and degradation) for Myc dependent and independent genes. The grey scale represents the starting level for each parameter in unstimulated cells.


A . Overlap between Myc ChIP - seq peaks . For each time - point indicated at bottom , of peaks overlapping over ≥ 1 bp with any other time - point ( Reference sample ) is reported . Peaks showed consistent distributions along time - course , almost those called in untreated being included in LPS - stimulated

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

A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samples


B , C . Distribution of Myc at promoters ( B ) distal sited ( C ) in LPS - treated B - ( 0 , 2 , 4 8h ) in B - in vivo , in either control c - mycwt / wt ( C ) , pre - tumoral Eµ - myc mice ( P ) or tumors ( T ) 4 [ ] . Each row represents a promoter - associated Myc peak within a 6 kb genomic interval ( centered on midpoint of peak ) . includes every annotated promoter in chromosome 1 ( as representative of whole genome ) that was called as Myc - associated by ChIP - seq in at least one sample . For same intervals , we also show distribution of H3K4me1 , H3K4me3 , H3K27ac in in vivo control sample , gene annotations ( exons in red , introns in pink ; + sense , - antisense strand ) .

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

B, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand).


D , E . Numbers of Myc peaks at promoters ( - 2kb , + 1kb ) ( D ) distal sites ( E ) at different time points after LPS stimulation . shadings mark subsets of peaks associated with H3K4me3 ( D ) or H3K4me1 ( E ) , with or without H3K27ac .

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

D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac.


F . of active promoters ( positive for H3K4me3 H3K27ac ) or distal sites ( H3K4me1 H3K27ac ) that overlap with at least a Myc peak at different time points after LPS stimulation ( 0 , 2 , 4 , 8h ) or in vivo ( C , P , T1 , T2 , T3 ) .

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F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3).


G . Quantitative showing Myc signal intensities ( read normalized by library ) at each bound region in chromosome 1 , ranked on signal in control sample ( 0h LPS ) .

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G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS).


H . of Myc peaks at promoters ( top ) or distal sites ( bottom ) that contain either canonical ( CACGTG ) or variant E - boxes ( CACGCG , CATGCG , CACGAG , CATGTG ) 83637 [ , , ] in a 200 bp window centered on peak summit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of Myc peaks at promoters (top) or distal sites (bottom) that contain either canonical (CACGTG) or variant E-boxes (CACGCG, CATGCG, CACGAG, CATGTG) 83637[,,] in a 200 bp window centered on the peak summit.


I . Myc enrichment by ChIP - seq at different time points after LPS treatment for peaks located either in promoters or distal sites containing indicated motifs . In boxplot , central tendency marks median , box ranges mark upper lower quartiles , whiskers extend to upper lower extreme datapoints which are no more than 1 . 5 times interquartile range from box . Myc ChIP - seq time - course was performed on a unique pool of , obtained from > 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.


A . Scatter plots correlating variations in Myc share at promoters ( averaged between 2 , 4 8h time points ) in synthesis ( at 8h ) , relative to untreated , for Myc - dependent independent genes . Spearman correlation between two parameters is reported .

O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plots correlating the variations in Myc share at promoters (averaged between the 2, 4 and 8h time points) and in RNA synthesis (at 8h), relative to untreated cells, for Myc-dependent and independent genes. The Spearman correlation between the two parameters is reported.


B . ability of Myc share in predicting transcriptional outcome can be represented in terms of a Receiver operating characteristic ( ROC ) curve , which represents Sensitivity ( true positive rate ) Specificity ( true negative rate ) of a predictor ( i . e . Myc share ) in discriminating between two classes ( i . e . up - down - regulated genes ) , using different thresholds . largest under curve ( AUC ) , largest is predictive power , in a scale from 0 . 5 ( random classification : grey ) to 1 ( perfect predictor ) . For each system , dot corresponds to variation of Myc at which promoters begin increasing their share of Myc binding .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic (ROC) curve, which represents the Sensitivity (true positive rate) and Specificity (true negative rate) of a predictor (i.e. Myc share) in discriminating between two classes (i.e. up- and down-regulated genes), using different thresholds. The largest the area under the curve (AUC), the largest is the predictive power, in a scale from 0.5 (random classification: grey line) to 1 (perfect predictor). For each system, the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding.


C . Fold - change in Myc at each bound promoter ( at 4h LPS , relative to time zero ) as a function of initial binding at time zero ( expressed as log2 of coverage in a 200 bp window around summit of peak ) . Peaks containing a canonical E - box are highlighted in black .

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

C. Fold-change in Myc levels at each bound promoter (at 4h LPS, relative to time zero) as a function of the initial binding intensity at time zero (expressed as log2 of the coverage in a 200 bp window around the summit of the peak). Peaks containing a canonical E-box are highlighted in black.


D . of Myc - bound promoters within indicated transcriptionally regulatory categories that contains a canonical or variant E - box . overall chi - square is 207 . 7 with a p - value < 0 . 00001 . contribution to chi - square of each category is reported above corresponding bar in plot .

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D. Percentage of Myc-bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E-box. The overall chi-square is 207.7 with a p-value <0.00001. The contribution to the chi-square of each category is reported above the corresponding bar in the plot.


B , C . Boxplots reporting LPS - induced changes ( Log2FC ) for each of four RNAPII kinetic rates in c - mycwt / wt ( wt ) c - myc ∆ / ∆ for Myc - dependent ( B ) Myc - independent ( C ) induced repressed genes . p - values obtained with two - sample Wilcoxon for comparison between c - mycwt / wt c - myc ∆ / ∆ are reported . RNAPII ChIP - seq time - course was performed on two biological replicates ( each on pools of from > 10 mice ) , reads were merged after sequencing .

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

B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing.


D , E . Heatmaps showing variations in RNAPII kinetic rates ( Log2FC ) on Myc - dependent ( D ) Myc - independent ( E ) genes during LPS time - course . grey scale represents starting level for each parameter in unstimulated . first column on left reports changes in Myc share for same genes . Genes were grouped using hierarchical clustering .

O O O O O 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

D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.


F . Boxplots reporting LPS - induced changes ( Log2FC ) for each of four RNAPII kinetic rates in c - mycwt / wt ( wt ) c - myc ∆ / ∆ for genes of clusters 1 9 . p - values obtained with two - sample Wilcoxon for comparison between c - mycwt / wt c - myc ∆ / ∆ are reported .

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

F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


G . Changes in Myc share in LPS treated versus untreated for different clusters of panel d . p - values obtained with two - sample Wilcoxon for comparison between c - mycwt / wt c - myc ∆ / ∆ are reported .

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

G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


( A ) FAK immunoblotting was performed on macrophages isolated from peritoneum of WT FAKΔmyeloid mice .

(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice.


( B ) WT FAK − / − PEMs were incubated with S . typhimurium ΔinvG for 0 - 5 hours before immunoblotting with indicated antibodies .

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(B) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-5 hours before immunoblotting with the indicated antibodies.


( C , E G ) WT PEMs expressing GFP - FAK were incubated with either ΔinvG Salmonella ( C ) , LPS - coated beads ( E ) or E . coli ( G ) for a total of 5 hours before by immunofluorescence . were co - stained with antibodies recognizing LAMP1 ( red ) . Dapi was used to visualize nuclei ( blue ) . Bars represent 10 µm . Arrowheads indicate or beads in enlarged panel where bars represent 2 µm . ( D , F H ) of LAMP1 - positive ( gray bars ) LAMP1 + GFP + ( black bars ) ΔinvG Salmonella ( D ) , LPS - coated beads ( F ) E . coli ( H ) was quantified . At least 100 were counted per condition ; N = 3 .

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(C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.


A ) TLR4 − / − PEMs were incubated with ΔinvG Salmonella for 0 - 5 hours before immunoblotting with indicated antibodies .

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A) TLR4−/− PEMs were incubated with ΔinvG Salmonella for 0-5 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with LPS - coated beads for 0 - 5 hours before immunoblotting for indicated antibodies .

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(B) WT PEMs were incubated with LPS-coated beads for 0-5 hours before immunoblotting for the indicated antibodies.


( C ) of phosphorylated were quantified by densitometry , normalized to amount of total present in each sample , expressed relative to basal level in uninfected . Values are means ± , N = 3 , * p < 0 . 05 . Dashed drawn at 1 indicates basal of activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( D ) WT PEMs were incubated with orgA : : tet ; spiA : : kan Salmonella deficient in both SPI1 SPI2 T3SSs ( ΔSPI1ΔSPI2 ) for 0 - 5 hours before immunoblotting with indicated antibodies .

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(D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0-5 hours before immunoblotting with the indicated antibodies.


( E ) WT PEMs expressing GFP - FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before by immunofluorescence . were co - stained with antibodies recognizing LAMP1 ( red ) . DAPI was used to visualize nuclei ( blue ) . Bars represent 10 µm or 2 µm in enlarged inset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.


( A ) WT FAK − / − PEMs were incubated for 0 - 5 hours with ΔinvG Salmonella before immunoblotting with indicated antibodies .

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(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies.


( B ) WT PEMs were pretreated for 1 hour with FAK - kinase inhibitor PF - 228 ( 0 . 5 µm ) before incubation with ΔinvG Salmonella for 5 hours . Lysates were immunoblotted with indicated antibodies .

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(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.


( C - D ) WT or FAK − / − macrophages were incubated with either ΔΣΠΙ1ΔΣΠΙ2 ( C ) or LPS - coated beads ( D ) for 0 - 5 hours before immunoblotting with indicated antibodies . In panels C D , WT PEMs were also incubated with ΔinvG ( 5 h ) for comparison of Akt activation . Vertical lines in panel D indicate non - contiguous lanes generated from a single exposure .

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(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.


E ) of phosphorylated were quantified by densitometry , normalized to amount of total present in each sample , expressed relative to basal level in uninfected . Values are means ± , N = 3 , * p < 0 . 05 . Dashed drawn at 1 indicates basal of activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( F ) WT PEMs expressing HA - Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours . Lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies before immunoblotting for FAK HA . In parallel , lysates were immunoblotted for FAK , HA ERK1 / 2 to control for level of expression .

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(F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression.