C - J : The FR2020 strain was used in subsequent experiments where pDC were either mock treated ( mock , grey ) , exposed to SARS - CoV - 2 at 1 MOI ( SARS - 2 , purple ) , TLR7 ( 2 . 5 μg / mL R837 , blue ) or TLR3 agonist ( 800 ng / mL poly ( I : C ) , pink ) . Supernatants were collected after 24 hrs and analyzed for type I IFNα ( C ) , IFNβ ( D ) , type II IFNγ ( E ) , type III IFNλ1 ( F ) , IL - 6 ( G ) , IL - 8 ( H ) , CXCL10 ( I ) and TNFα ( J ) expression by ELISA . Data information : Bars represent mean values with standard error of the mean and symbols represent individual pDC donors ( n = 3 - 4 ) . Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point - matched mock condition * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 .

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

C- J: The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. Data information: Bars represent mean values with standard error of the mean and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition *<p0.05, **<p0.01 ***<p0.001.


Using CRISPR / Cas9 , MyD88 knock - out ( KO ) and AAVS1KO ( control ) pDCs were generated . MyD88 protein levels in KO and control pDCs were analyzed by western

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

Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting


C - D : MyD88KO and control pDCs were either mock treated ( mock ) or exposed to SARS - CoV - 2 ( SARS - 2 , 1 MOI ) , supernatants were collected at indicated time points and analyzed for type I IFNα ( C ) and CXCL10 ( D ) . Done with n = 4 Data information : Bars represent mean values and equal symbols represent equal donors . Statistical significance was determined using Mann - Whitney one - tailed T test in ( C ) as MyD88 - KO data had no variance and values of 0 . For ( D ) a Mann - Whitney two - tailed T test was used for comparison between conditions of mock and MyD88 KO donors .

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C-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4 Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors.


E - F : Type I IFNα production was then determined in cell culture supernatant from SARS - CoV - 2 exposed TRIFKO or TRIG + MyD88KO ( E ) and RIG - IKO or RIG - I + MyD88KO ( F ) pDCs . Done with n =

O O O O O O O O O O O O O O O O O O O O O O O O O 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: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2


( A ) Representative micro - CT images and H & E staining of trabecular bone from the femoral metaphysis of 6 - week - old male Sp7 - Cre ; Cdc20f / f and littermate control mice . Scale bar , 500 μm . ( B ) Histomorphometric analyses of 6 - week - old male femurs . ( n = 6 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (B) Histomorphometric analyses of 6-week-old male femurs. (n=6)


( C ) Representative micro - CT images and H & E staining of trabecular bone from the femoral metaphysis of 6 - week - old female Sp7 - Cre ; Cdc20f / f and littermate control mice . Scale bar , 500 μm . ( D ) Histomorphometric analyses of 6 - week - old female femurs . ( n = 6 )

(C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (D) Histomorphometric analyses of 6-week-old female femurs. (n=6)


( E ) Representative micro - CT images of tibial cortical bone defects in Sp7 - Cre ; Cdc20f / f and littermate control mice . The green lines show the position of the original defect position . The green circle represents the regenerated bone . Scale bar , 500 μm . ( F ) Histomorphometric analysis of the regenerated bone in tibial cortical gaps . ( n = 5 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm. (F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)


( B , C ) The ALP staining ( B ) and ALP quantification ( C ) of Cdc20f / f and Sp7 - Cre ; Cdc20f / f BMSCs after 7 days osteogenic differentiation . ( n = 6 )

O O O O O O O O O O O O O O O O O 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) The ALP staining (B) and ALP quantification (C) of Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs after 7 days osteogenic differentiation. (n=6)


( E , F ) The ALP staining ( E ) and ALP quantification ( F ) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

(E, F) The ALP staining (E) and ALP quantification (F) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( H , I ) The ALP staining ( H ) and ALP quantification ( I ) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

O O O O O O O O O O O O O O O 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) The ALP staining (H) and ALP quantification (I) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( E ) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20 . The cytoplasmic and nuclear protein of overexpressed Myc - CDC20 and Vector HEK293T cells were extracted after TNF - α treatment for 30 minutes .

O O O O O O O O O O O O O O O O O O O O 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) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20. The cytoplasmic and nuclear protein of overexpressed Myc-CDC20 and Vector HEK293T cells were extracted after TNF-α treatment for 30 minutes.


( G ) Western blot analyses of the degradation of p65 protein after the overexpression of Myc - CDC20 . hBMSCs were transfected with Vector and Myc - CDC20 plasmids for 36 h , cells were treated with or without 10 μM MG132 for 6 h before collected .

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

(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.


( E , F ) Co - immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells .

(E, F) Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells.


( G ) Co - immunoprecipitation of purified GST - CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST - CDC20 . Whole cell lysates ( WCL ) were subjected to perform GST pull - down analyses with purified GST - CDC20 and GST as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation of purified GST-CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST-CDC20. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-CDC20 and GST as a negative control.


( G , H ) Co - immunoprecipitation of ectopically expressed Myc - CDC20 171 - 499 with ectopically expressed Flag - p65 1 - 310 fragment in HEK293T cells .

O O O O O O O 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) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.


( C ) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells .

(C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells.


( F ) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA - APC11 plasmids .

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(F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids.


( H , I ) The ALP staining ( H ) and ALP quantification ( I ) of NC and APC11si hBMSCs after 7 days osteogenic differentiation . ( n = 6 )

(H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)


( A ) Immunoblot of Flag - p65 - linked ubiquitination promoted by Myc - CDC20 . HEK293T cells were co - transfected with Flag - p65 , HA - Ubiquitin , with or without Myc - CDC20 or Vector plasmids for 36 h . Transfected cells were then treated with 10 μM MG132 for 6 h before collection . ( B ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 . HEK293T cells were transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 or Vector plasmids for 36 h . Transfected cells were then treated with 10 μM MG132 for 6 h before collection .

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

(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.


( F ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 in NC and CDC20sh cells . NC or CDC20sh HEK293T cells were co - transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 and Vector plasmids . Transfected cells were treated with 10 μM MG132 for 6 h before collection .

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

(F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.


( A ) Representative micro - CT images of trabecular bone from the femoral metaphysis of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , and Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . Scale bar , 500 μm . ( B ) Histomorphometric analyses of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , and Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . ( n = 6 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm. (B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6)


( C , D ) The ALP staining ( C ) and ALP quantification ( D ) of BMSCs from Cdc20f / f mice and Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si . ( n = 5 )

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5)


( G ) The expression of RUNX2 in BMSCs from Cdc20f / f mice and Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses .

(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , WDR74 and HAUS5 promoters for IgG and DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) .

O O O O O O O O O O O O O O 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-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , COX14 , HLA - E and PSMB8 promoters for IgG and DVL3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) .

O O O O O O O O O O O O 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) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( E ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , WDR74 , HCG15 , POU2F3 , SNORD3B , APOC1P1 , HLA - A , TAP1 , PSMD8 , PDG1 and JUNB genes in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , PCAT6 , ZNF672 , SSC5D , APOC1P1 , HLA - A , HLA - F and TAP1 genes in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . D

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , KDM5B , APOC1P1 , and PSMB8 promoters for IgG and H3K4me3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) .

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , PCAT6 , APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) .

O O O O O O O O O O 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) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , and PSMB8 promoters for IgG and KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) .

O O O O O O O O O O O O O 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-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MDA - MB - 468 for the PRDM10 , TRIM26 , BTN2A1 , PNRC2 , and MR1 genes , visualized by IGV . The green rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MDA - MB - 468 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.


( E ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MCF7 for the WDR74 , YWHAQ , CDNF / HSPA14 , BTN2A1 , and MR1 genes , visualized by IGV . The turquoise and purple rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MCF7 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.


( D ) RT - qPCR - based analysis of expression changes of DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( E ) RT - qPCR - based analysis of expression changes of KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of DVL3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( G ) RT - qPCR - based analysis of expression changes of KMT2D in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( H

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H


( H ) Immunofluorescence of KMT2D localization in MDA - MB - 468 cells . The cells were probed with KMT2D antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of KMT2D ( red ) and nuclear staining ( blue ) is shown as KMT2D / DAPI , merge of KMT2D ( red ) and actin ( green ) is shown as KMT2D / ACTIN , and , merge of KMT2D ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for NTC , shA DVL3 and shB DVL3 cells . Scale bar :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm


( C ) IVIS images showing representative tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( D ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice , Average luminescence was used to measure volume of tumor ; n ≥ 9 . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. (


( G ) IVIS images showing representative tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( H ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Average luminescence was used to measure volume of tumor . n = 7 .

(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.


( A ) Immunofluorescence of HA - tagged DVL3 localization in MDA - MB - 468 cells . The cells were probed with an HA antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of actin ( green ) and nuclear staining ( blue ) is shown as ACTIN / DAPI , merge of DVL3 ( red ) and nuclear staining ( blue ) is shown as HA / DAPI , merge of DVL3 ( red ) and actin ( green ) is shown as HA / ACTIN , and , merge of DVL3 ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for empty vector ( EV ) , HA - DVL3 - WT and HA - DVL3 - NESm transient transfected cells . Scale bar : 10µm .

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(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.


( B ) RT - qPCR - based analysis of expression changes of DVL3 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( C ) RT - qPCR - based analysis of expression changes of KMT2D in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( D ) RT - qPCR - based analysis of expression changes of ARAP1 , RFX5 , GBAT2 , WDR74 , SNORD3D , HAUS5 , TAP1 , HLA - A , PDG1 , and , MR1 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( E

(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E


A Dose - response viability curves in presence of increasing concentrations of IMT1 for one - week , viable cells were counted and normalized to DMSO - treated controls . Data are expressed as mean ± SD of n = 3 independent experiments with four technical replicates . IC50 values were calculated with non - linear least square fit , RKO : 521 . 8 nM , MiaPaCa - 2 : 291 . 4 nM and HeLa : 29 . 9 nM .

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A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.


B Mitochondrial transcript levels measured by qRT - PCR in RKO cells after 96 hours of 1 μM IMT1 treatment . Data are relative to DMSO - treated controls and are expressed as mean ± SD of n = 4 independent experiments . Ordinary one - way ANOVA was used for comparisons to DMSO - treated controls ( RNR1 , RNR2 , MT - COX2 , MT - ATP6 , MT - ND1 : * * * p < 0 , 0001 ) .

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B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).


D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours . Data are plotted as average log2 - fold change versus log10 of adjusted p - value of DMSO - treated controls ; mitochondrial protein are highlighted in red .

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D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.


F CRISPR guides enriched ( positive hits ) in IMT1 - treated RKO cells plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value . mTORC1 and VHL pathway - related genes are reported in green and blue , respectively .

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F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.


B - D Viable cells count in presence of DMSO , IMT1 alone or in combination with rapamycin in RKO ( B ) , MiaPaCa - 2 ( C ) and HeLa ( D ) cells . Data are expressed as mean values ± SD of n = 6 independent experiments each including four technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA test . Paired IMT1 vs . IMT1 + RAPA comparisons : RKO : * * * p < 0 , 0001 , MiaPaCa - 2 : * * p = 0 , 0014 , HeLa : * p = 0 , 0238 .

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B-D Viable cells count in presence of DMSO, IMT1 alone or in combination with rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.


F - H Viability assessment of RKO ( F ) , MiaPaCa - 2 ( G ) and HeLa ( H ) cells treated with DMSO , IMT1 alone and in combination with FG4592 . Data are expressed as mean values ± SD of n = 6 , 5 , 6 independent experiments for RKO , HeLa and MiaPaCa - 2 , respectively . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs IMT1 + FG4592 comparisons : RKO : * * p < 0 , 0028 , MiaPaCa - 2 : * * p = 0 , 0011 , HeLa : * p = 0 , 0029 .

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F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.


C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO , IMT1 , rapamycin , rapamycin with IMT1 , FG4592 and FG4592 with IMT1 . ACTINB is shown as loading control .

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C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control.


F , G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function . RKO cells were treated with DMSO , rapamycin ( F ) or FG4592 ( G ) in presence or absence of IMT1 . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test . DMSO - treated controls vs : IMT1 : p = 0 , 0008 ; RAPA : p = 0 , 1578 ; RAPA + IMT1 : p = 0 , 0034 ; FG4592 : p = 0 , 872 ; FG4592 + IMT1 : p = 0 , 0029 . Paired t - test of IMT1 vs RAPA + IMT1 : p = 0 , 034 ; IMT1 vs FG4592 + IMT1 : non - significant .

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F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.


A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells . The graph shows mean values ± SEM of n = 3 independent experiments .

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A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.


B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO ( left ) and IMT1 - resistant RKO cells ( right ) . The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line ; GAPDH is shown as loading control .

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B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.


G Relative mtDNA copy number measured in RKO and IMT1 - resistant cells treated with IMT1 for 96 hours . Data are expressed as average from n = 3 experiments ± SEM . Statistical significance was calculated with one - way ANOVA test . RKO + IMT1 vs Resistant + IMT1 : MT - ND1 * * p = 0 , 0035 , MT - ATP6 * * * p = 0 , 0002 , MT - CYTB * * p = 0 , 0028 .

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G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.


H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1 - resistant RKO cells treated with DMSO or IMT1 . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test . RKO DMSO vs RKO + IMT1 : p = 0 , 0008 ; RKO DMSO vs resistant DMSO : p = 0 , 0343 ; RKO DMSO vs resistant + IMT1 : p = 0 , 0092 . Paired t - test of RKO + IMT1 vs resistant + IMT1 : p = 0 , 135 .

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H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.


A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1 - treated RKO and IMT1 - resistant cells . Data are expressed as the mean ± SEM of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA test for multiple comparisons . RKO DMSO vs RKO + IMT1 : * * p = 0 , 0019 ; RKO + IMT1 vs resistant + IMT : * p = 0 , 0214 ; RKO DMSO vs resistant DMSO : non - significant .

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A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.


D , E IMT1 extracellular ( D ) and extracellular ( E ) concentrations measured in the medium at 0 , 2 and 24 hours of IMT1 treatment in RKO and IMT1 - resistant RKO cells . Mean values ± SEM of n = 3 experiments .

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D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.


B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours ( r : sample correlation coefficient ) . Data are expressed as average log2 - fold change of controls ( DMSO - treated RKO cells ) . Mitochondrial proteins are reported in red .

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B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.


A Depleted CRISPR guides ( negative hits ) in IMT1 - treated RKO cells plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value .

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A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.


C Representative images and related plot of IMT1 - resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks ( IMT1 : 1 μM , CAP : 1 μg / ml ) ; Scale bar : 1mm . Data show the mean values ± SD of n = 4 experiments . Statistical significance was calculated with paired t - test : IMT1 vs IMT1 + CAP : * p = 0 , 0242 .

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C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.


G Viable cells counts of IMT1 - resistant RKO cells treated for one - week with either DMSO or IMT1 in presence of controls ( control # 1 , # 2 ) or TFAM ( TFAM # 1 , TFAM # 2 ) siRNAs . Data are expressed as mean values ± SD of n = 5 independent experiments each including six technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA test . Controls + IMT1 vs TFAM # 1 + IMT1 : p = 0 , 2148 ; Controls + IMT1 vs TFAM # 2 + IMT1 : * p = 0 , 0415 .

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G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.


( A ) RAW 264 . 7 macrophages infected with heat killed ( top panels ) or live ( bottom panels ) S . aureus USA300 ( in red ) were imaged by DIC time - lapse microscopy at 6 h post - infection . The images on the left are of time zero in the image sequence and show the presence of S . aureus . The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads . Candidate pinosomes are denoted by yellow arrows .

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(A) RAW 264.7 macrophages infected with heat killed (top panels) or live (bottom panels) S. aureus USA300 (in red) were imaged by DIC time-lapse microscopy at 6 h post-infection. The images on the left are of time zero in the image sequence and show the presence of S. aureus. The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads. Candidate pinosomes are denoted by yellow arrows.


( B ) RAW 264 . 7 macrophages expressing the lipid biosensor for PI ( 4 , 5 ) P2 , PH - Plcδ - RFP ( in red ) , were left uninfected or were infected with live GFP - expressing S . aureus USA300 ( in green ) for 8 h prior to fixation . The macrophage plasmalemma and extracellular bacteria are indicated by Cy5 - conjugated wheat germ agglutinin ( WGA , in blue ) . As a control , to demonstrate displacement of PH - Plcδ - RFP from the macrophage membrane , transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation . These images are representative of three independent experiments .

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(B) RAW 264.7 macrophages expressing the lipid biosensor for PI(4,5)P2, PH-Plcδ-RFP (in red), were left uninfected or were infected with live GFP-expressing S. aureus USA300 (in green) for 8 h prior to fixation. The macrophage plasmalemma and extracellular bacteria are indicated by Cy5-conjugated wheat germ agglutinin (WGA, in blue). As a control, to demonstrate displacement of PH-Plcδ-RFP from the macrophage membrane, transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation. These images are representative of three independent experiments.


( C , D ) Internalization of FITC - Dextran by uninfected ( top row ) and S . aureus ( in blue ) - infected ( bottom row ) RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . The quantitation of FITC - dextran uptake by either uninfected or infected RAW 264 . 7 and primary bone marrow derived macrophages is shown in ( D ) . For each experiment , macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u . ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

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(C, D) Internalization of FITC-Dextran by uninfected (top row) and S. aureus (in blue)-infected (bottom row) RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. The quantitation of FITC-dextran uptake by either uninfected or infected RAW 264.7 and primary bone marrow derived macrophages is shown in (D). For each experiment, macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( E ) Potential for co - localization of FITC - dextran ( in green ) with phagocytosed S . aureus expressing mCherry ( in red ) . The micrographs depict representative macrophages infected with S . aureus at 10 h post - infection and the white arrows point to mCherry positive bacteria that are co - localizing with dextran . Macrophages were treated with 1 μM concanamycin A prior to imaging live .

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(E) Potential for co-localization of FITC-dextran (in green) with phagocytosed S. aureus expressing mCherry (in red). The micrographs depict representative macrophages infected with S. aureus at 10 h post-infection and the white arrows point to mCherry positive bacteria that are co-localizing with dextran. Macrophages were treated with 1 μM concanamycin A prior to imaging live.


( A ) Internalization and processing of DQ ™ Green BSA by uninfected and S . aureus ( in blue ) infected RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . ( B ) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S . aureus ( in blue ) infected RAW 264 . 7 . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Internalization and processing of DQ™ Green BSA by uninfected and S. aureus (in blue) infected RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. (B) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S. aureus (in blue) infected RAW 264.7. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( C ) Fluorescence - based proliferation assays were performed in conjunction with the probe DQ ™ Green BSA . Processed DQ ™ Green BSA could be found co - localizing with intracellular mCherry - expressing S . aureus ( in red ) and with intracellular S . aureus has replicated ( i . e . is proliferation dye negative ) at 10h post - infection as depicted here . Data information : Scale bars equal 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence-based proliferation assays were performed in conjunction with the probe DQ™ Green BSA. Processed DQ™ Green BSA could be found co-localizing with intracellular mCherry-expressing S. aureus (in red) and with intracellular S. aureus has replicated (i.e. is proliferation dye negative) at 10h post-infection as depicted here. Data information: Scale bars equal 10 μm.


( A ) Uptake of Alexa ® Fluor647 - conjugated transferrin by RAW 264 . 7 macrophages in the presence and absence of PIK - III and Dynasore . The fluorescence micrographs show transferrin ( in blue ) and the macrophage plasmalemma demarcated by TMR - conjugated wheat germ agglutinin ( in red ) . The dashed line indicates the region of the cell for which the line scan analysis , shown graphically on the right , was done . These micrographs are representative images obtained from three independent experiments . Line scan analysis was performed to indicate the distribution of fluorescent transferrin ( blue line ) relative to the plasmalemma ( red line ) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs . Scale bars equal 10 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O

(A) Uptake of Alexa® Fluor647-conjugated transferrin by RAW 264.7 macrophages in the presence and absence of PIK-III and Dynasore. The fluorescence micrographs show transferrin (in blue) and the macrophage plasmalemma demarcated by TMR-conjugated wheat germ agglutinin (in red). The dashed line indicates the region of the cell for which the line scan analysis, shown graphically on the right, was done. These micrographs are representative images obtained from three independent experiments. Line scan analysis was performed to indicate the distribution of fluorescent transferrin (blue line) relative to the plasmalemma (red line) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs. Scale bars equal 10 μm.


( B ) RAW macrophages were pre - treated with vehicle ( DMSO ) or the vps34 inhibitor PIK - III prior to the addition of 10 kDa FITC - dextran ( 100 µg / mL ) for 2 hrs . Prior to imaging live , macrophages were also stained with Cy5 - conjugated WGA . The representative micrographs depict inhibited dextran uptake indicating PIK - III treatment can perturb fluid phase uptake . Scale bars equal 10 µm .

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

(B) RAW macrophages were pre-treated with vehicle (DMSO) or the vps34 inhibitor PIK-III prior to the addition of 10 kDa FITC-dextran (100 µg/mL) for 2 hrs. Prior to imaging live, macrophages were also stained with Cy5-conjugated WGA. The representative micrographs depict inhibited dextran uptake indicating PIK-III treatment can perturb fluid phase uptake. Scale bars equal 10 µm.


( C ) The intensity of dextran fluorescence was quantified for RAW cells pre - treated with vehicle , PIK - III ( 10 µM ) , Dynasore ( 100 µM ) or PIK - III and Dynasore ( 10 and 100 µM ) respectively . The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments . * indicates p < 0 . 05 , as determined by an ordinary one - way ANOVA with a Bonferroni multiple comparisons test .

O O O O O O B-MEASURED_VAR 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 O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The intensity of dextran fluorescence was quantified for RAW cells pre-treated with vehicle, PIK-III (10 µM), Dynasore (100 µM) or PIK-III and Dynasore (10 and 100 µM) respectively. The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments. * indicates p<0.05, as determined by an ordinary one-way ANOVA with a Bonferroni multiple comparisons test.


( A ) The effects of prolonged exposure to gentamicin ( 300 µg / mL ) or vancomycin ( 300 µg / mL ) on the survival of phagocytosed S . aureus USA300 in RAW macrophages is shown . Depicted above the graph is a timeline detailing the treatment regime . The data are presented as the mean fold change in CFU / mL + / - standard deviation for 15 biological replicates of S . aureus , represented by each symbol , from five independent experiments . For each replicate the fold change was calculated by dividing the bacterial count at 10 h post - infection by the counts at 1 . 5 h post - infection . The dashed line indicates a fold change value equal to 1 ( i . e . no change in bacterial burden ) . * * p < 0 . 01 and statistical significance was determined by a one - way ANOVA with a Dunnett ' s post - hoc test .

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

(A) The effects of prolonged exposure to gentamicin (300 µg/mL) or vancomycin (300 µg/mL) on the survival of phagocytosed S. aureus USA300 in RAW macrophages is shown. Depicted above the graph is a timeline detailing the treatment regime. The data are presented as the mean fold change in CFU/mL +/- standard deviation for 15 biological replicates of S. aureus, represented by each symbol, from five independent experiments. For each replicate the fold change was calculated by dividing the bacterial count at 10 h post-infection by the counts at 1.5 h post-infection. The dashed line indicates a fold change value equal to 1 (i.e. no change in bacterial burden). **p<0.01 and statistical significance was determined by a one-way ANOVA with a Dunnett's post-hoc test.


( B ) The ability to protect S . aureus USA300 from intracellular killing by gentamicin is shown . Here , experiments were performed as in ( A ) except during the initial gentamicin treatment ( 100 μg / mL for 1h ) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK - III / Dynasore to inhibit fluid phase uptake . At 1 . 5 h post - infection cells were washed and treated with gentamicin ( 300 µg / mL ) in the presence of either DMSO ( vehicle ) or PIK - III + Dynasore which were maintained throughout the experiment . The data are presented as the mean number of surviving bacteria in CFU / mL at 10 h post - infection + / - standard deviation . These data derive from a total of 15 biological replicates of S . aureus from at least three independent experiments . * * * p < 0 . 001 and statistical significance was determined by an unpaired two - tailed t - test with a Welch ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 to protect S. aureus USA300 from intracellular killing by gentamicin is shown. Here, experiments were performed as in (A) except during the initial gentamicin treatment (100 μg/mL for 1h) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK-III/Dynasore to inhibit fluid phase uptake. At 1.5 h post-infection cells were washed and treated with gentamicin (300 µg/mL) in the presence of either DMSO (vehicle) or PIK-III + Dynasore which were maintained throughout the experiment. The data are presented as the mean number of surviving bacteria in CFU/mL at 10 h post-infection +/- standard deviation. These data derive from a total of 15 biological replicates of S. aureus from at least three independent experiments. *** p<0.001 and statistical significance was determined by an unpaired two-tailed t-test with a Welch's correction.


( C ) The effect of PIK - III / Dynasore treatment on the ability of S . aureus USA300 to replicate within RAW macrophages is shown . After gentamicin treatment at 1 . 5 h , RAW 264 . 7 macrophages having phagocytosed S . aureus were lysed and plated to determine the number of gentamicin protected bacteria . Alternatively , infected samples treated with either DMSO as a vehicle control or with PIK - III / Dynasore which was added at 1 . 5h post - infection after gentamicin . At 10 h post - infection macrophages exposed to DMSO or inhibitors , that were maintained throughout the experiment , were lysed and plated to determine the bacterial burden . The data shown are the mean fold change in CFU / mL at 10 h where the data are normalized to the bacterial burden obtained at 1 . 5 h . Each symbol represents a biological replicate ( n = 8 ) of S . aureus and derive from three independent experiments . The data presented are the mean ± standard deviation . * indicates p < 0 . 05 as determine by an unpaired two - tailed t - test with a Welch ' s correction .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of PIK-III/Dynasore treatment on the ability of S. aureus USA300 to replicate within RAW macrophages is shown. After gentamicin treatment at 1.5 h, RAW 264.7 macrophages having phagocytosed S. aureus were lysed and plated to determine the number of gentamicin protected bacteria. Alternatively, infected samples treated with either DMSO as a vehicle control or with PIK-III/Dynasore which was added at 1.5h post-infection after gentamicin. At 10 h post-infection macrophages exposed to DMSO or inhibitors, that were maintained throughout the experiment, were lysed and plated to determine the bacterial burden. The data shown are the mean fold change in CFU/mL at 10 h where the data are normalized to the bacterial burden obtained at 1.5 h. Each symbol represents a biological replicate (n=8) of S. aureus and derive from three independent experiments. The data presented are the mean ± standard deviation. * indicates p<0.05 as determine by an unpaired two-tailed t-test with a Welch's correction.


( D ) The effect of PIK - III + Dynasore or DMSO on the growth of S . aureus USA300 in RPMI with 5 % ( v / v ) FBS is shown . The data are the mean OD600nm + / - standard deviation of four biological replicates of S . aureus for each condition and are representative of three independent experiments .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of PIK-III + Dynasore or DMSO on the growth of S. aureus USA300 in RPMI with 5% (v/v) FBS is shown. The data are the mean OD600nm +/- standard deviation of four biological replicates of S. aureus for each condition and are representative of three independent experiments.


( E ) Analysis of the effects of PIK - III / Dynasore treatment on S . aureus replication using a fluorescence based bacterial proliferation assay is shown . The micrographs depict S . aureus USA300 expressing GFP that were co - labelled with proliferation dye . The top row of micrographs depict vehicle treated macrophages where GFP - positive , yet proliferation dye - negative bacteria were present . The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive . The macrophage plasmalemma and extracellular bacteria are demarcated by TMR - WGA ( in red ) . Images were acquired at 10 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of the effects of PIK-III/Dynasore treatment on S. aureus replication using a fluorescence based bacterial proliferation assay is shown. The micrographs depict S. aureus USA300 expressing GFP that were co-labelled with proliferation dye. The top row of micrographs depict vehicle treated macrophages where GFP-positive, yet proliferation dye-negative bacteria were present. The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive. The macrophage plasmalemma and extracellular bacteria are demarcated by TMR-WGA (in red). Images were acquired at 10 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm.


( F ) The normalized fraction of macrophages containing replicating bacteria is shown . The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition . Each symbol represents a biological replicate ( n = 6 ) of S . aureus and derive from three independent experiments . * * * * p < 0 . 0001 and statistical significance was determined by an unpaired two - tailed t - test using a Welch ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The normalized fraction of macrophages containing replicating bacteria is shown. The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition. Each symbol represents a biological replicate (n=6) of S. aureus and derive from three independent experiments. ****p<0.0001 and statistical significance was determined by an unpaired two-tailed t-test using a Welch's correction.


( A ) Growth of wild - type S . aureus USA300 or a purK mutant . The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI . Additives are indicated on the x axis and were either 5 % ( v / v ) FBS , 50 μM IMP , 10 μM PIK - III and 100 μM Dynasore or an equivalent volume of DMSO . Each symbol represents a biological replicate of S . aureus and the data derive from three independent experiments . Statistical significance was determined by a one - way ANOVA with a Holm - Sidak ' s multiple comparison test . n . s . indicates not significant , * * * indicates p < 0 . 0001 .

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

(A) Growth of wild-type S. aureus USA300 or a purK mutant. The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI. Additives are indicated on the x axis and were either 5% (v/v) FBS, 50 μM IMP, 10 μM PIK-III and 100 μM Dynasore or an equivalent volume of DMSO. Each symbol represents a biological replicate of S. aureus and the data derive from three independent experiments. Statistical significance was determined by a one-way ANOVA with a Holm-Sidak's multiple comparison test. n.s. indicates not significant, *** indicates p<0.0001.


( B ) The graph depicts the fold change in bacterial burden for RAW 264 . 7 macrophages infected with purK S . aureus . Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK - III and Dynasore which were maintained throughout the infection . The data are the mean ± standard deviation of the burden at 10 h post - infection normalized to the burden at 1 . 5h post - infection prior to the addition of IMP . Each symbol represents a single biological replicate of S . aureus and derive from three independent experiments . Significance was determined by one - way ANOVA with a Turkeys multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 .

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

(B) The graph depicts the fold change in bacterial burden for RAW 264.7 macrophages infected with purK S. aureus. Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK-III and Dynasore which were maintained throughout the infection. The data are the mean ± standard deviation of the burden at 10 h post-infection normalized to the burden at 1.5h post-infection prior to the addition of IMP. Each symbol represents a single biological replicate of S. aureus and derive from three independent experiments. Significance was determined by one-way ANOVA with a Turkeys multiple comparison test. *p<0.05, **p<0.01.


( C , D ) The ability of purK S . aureus to grow in an IMP - dependent manner within primary bone marrow - derived macrophages is shown . ( C ) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non - replicating bacteria are both GFP and proliferation dye positive . In contrast , replicating bacteria ( white arrows ) are only GFP positive . The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S . aureus in the presence of 50 μM IMP . Images were acquired at 12 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm . ( D ) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs . Each data point represents a biological replicate ( n = 8 ) and derived from three independent experiments . The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition . * * indicates p < 0 . 01 as determined by an unpaired two - tailed t - test with a Wilcoxon matched pairs test .

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 B-CONTROLLED_VAR 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 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

(C, D) The ability of purK S. aureus to grow in an IMP-dependent manner within primary bone marrow-derived macrophages is shown. (C) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non-replicating bacteria are both GFP and proliferation dye positive. In contrast, replicating bacteria (white arrows) are only GFP positive. The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S. aureus in the presence of 50 μM IMP. Images were acquired at 12 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (D) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs. Each data point represents a biological replicate (n=8) and derived from three independent experiments. The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition. ** indicates p<0.01 as determined by an unpaired two-tailed t-test with a Wilcoxon matched pairs test.


( D , E ) Imaging of luciferase activity in the liver of HIF1aAlbKO ( D ) and HIF2aAlbKO ( E ) mice after a hydrodynamic tail vein injection with a HRE - luc reporter plasmid or control ( PBS ) according to body weight followed by 6h normoxia or hypoxia . ( F , G ) Bioluminescent photon counts normalized to the PBS control group in liver of ( F ) HIF1aAlbKO and ( G ) HIF2aALbKO mice and their wild - type littermates 6h after normoxia of hypoxia . Data information : All bars represent mean ± SEM . Each individual data point represent individual mice . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 . N = normoxia , H = hypoxia . Signal around the perimeter of the Petri dishes represent aspecific luciferase signal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Imaging of luciferase activity in the liver of HIF1aAlbKO (D) and HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or hypoxia. (F, G) Bioluminescent photon counts normalized to the PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia of hypoxia. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal.


( E ) Scatter plot showing log fold change ( LFC ) of all DEX - upregulated genes ( LFC > 1 and P ≤ 0 . 05 ) and DEX - downregulated genes ( LFC < - 1 and P ≤ 0 . 05 ) in normoxia vs hypoxia after 24h . The black line represents the diagonal , and the red line represents the real slope ± standard error of the data as analysed by linear regression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot showing log fold change (LFC) of all DEX-upregulated genes (LFC > 1 and P ≤ 0.05) and DEX-downregulated genes (LFC < -1 and P ≤ 0.05) in normoxia vs hypoxia after 24h. The black line represents the diagonal, and the red line represents the real slope ± standard error of the data as analysed by linear regression.


( F , G ) Examples of GR - responsive genes based on the RNA - seq data 6h ( F ) and 24h ( G ) after normoxia or hypoxia and DEX stimulation . Data information : All bars represent mean ± SEM . N = 3 per group . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 .

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

(F, G) Examples of GR-responsive genes based on the RNA-seq data 6h (F) and 24h (G) after normoxia or hypoxia and DEX stimulation. Data information: All bars represent mean ± SEM. N=3 per group. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01.


( H ChIP - seq on liver derived from mice which were subjected to hypoxia ( 6h and 24h ) , followed by DEX ( 10 mg / kg ) injection and 2h later sacrificed for liver isolation . ( H ) Box plots showing the LFC of genes responsive to DEX in both normoxia or hypoxia ( 6h and 24h ) . The central band represents the median . The box ranges from the first quartile ( Q1 ) to the third quartile ( Q3 ) which represents the interquartile range ( IQR = Q3 - Q1 ) and covers the central 50 % of the data . The whiskers illustrate the minimum ( Q1 - 1 . 5 * IQR ) and maximum ( Q3 + 1 . 5 * IQR ) of the data . Outliers are shown as dots . N = 3 biological replicates per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (H) Box plots showing the LFC of genes responsive to DEX in both normoxia or hypoxia (6h and 24h). The central band represents the median. The box ranges from the first quartile (Q1) to the third quartile (Q3) which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition.


Female C57BL / 6J mice were put in normoxia or hypoxia for 6h or 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) and livers were isolated for genome - wide transcriptomics via RNA - seq . N = 3 per group for a single RNA - seq . ( A , B ) Examples of DEX - responsive genes in hypoxia 6h ( A ) and 24h ( B ) which were not induced by DEX in normoxia . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

Female C57BL/6J mice were put in normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single RNA-seq. (A, B) Examples of DEX-responsive genes in hypoxia 6h (A) and 24h (B) which were not induced by DEX in normoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.


ChIP - seq on liver derived from mice which were subjected to hypoxia ( 6h and 24h ) , followed by DEX ( 10 mg / kg ) injection and 2h later sacrificed for liver isolation . ( E ) Examples of specific GR DNA - binding peaks , associated with the DEX - induced gene Slc22a5 in hypoxia ( 6h and 24h ) .

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

ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (E) Examples of specific GR DNA-binding peaks, associated with the DEX-induced gene Slc22a5 in hypoxia (6h and 24h).


( F ) HIF1aHIF2afl / fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX . Slc25a30 expression was measured in the liver via RT - qPCR . N = 3 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(F) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX. Slc25a30 expression was measured in the liver via RT-qPCR. N=3 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.


( C ) Heatmap representing log2 values of shared stress GRE genes ( counts ) induced by hypoxia 6h and 24h and induced by DEX in normoxia ( LFC > 1 and P ≤ 0 . 05 ) .

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

(C) Heatmap representing log2 values of shared stress GRE genes (counts) induced by hypoxia 6h and 24h and induced by DEX in normoxia (LFC > 1 and P ≤ 0.05).


( D - G ) Female C57BL / 6J and ADX mice were put in normoxia or hypoxia for 6h or 24h , n = 6 / group , two independent experiments . ( D , E ) Confirmation of RNA - seq data via RT - qPCR [ 6h ( D ) or 24h ( E ) ] . ( F , G ) Plasma GC concentration [ 6h ( F ) or 24h ( G ) ] . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(D-G) Female C57BL/6J and ADX mice were put in normoxia or hypoxia for 6h or 24h, n=6/group, two independent experiments. (D, E) Confirmation of RNA-seq data via RT-qPCR [6h (D) or 24h (E)]. (F, G) Plasma GC concentration [6h (F) or 24h (G)]. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( M ) HIF1aHIF2afl / fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h . Stress GRE genes were measured in the liver via RT - qPCR . N = 4 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(M) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h. Stress GRE genes were measured in the liver via RT-qPCR. N=4 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( N ) Expression levels of DEX responsive genes in normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during hypoxia ( 24h ) via RT - qPCR . N = 3 - 4 per group , one experiment . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(N) Expression levels of DEX responsive genes in normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during hypoxia (24h) via RT-qPCR. N=3-4 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( D ) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control . HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels . P - values were calculated using Mann - Whitney test . N = 5 biological replicates per group . N = normoxia , H = hypoxia . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control. HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels. P-values were calculated using Mann-Whitney test. N=5 biological replicates per group. N = normoxia, H = hypoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, **P<0.01, *P≤0.05.


( A ) Female C57BL / 6J and ADX mice were put in normoxia and hypoxia for 6h and 24h . Plasma was isolated for metabolomics analysis . The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL / 6J mice ( C57BL / 6J P ≤ 0 . 05 and ADX P > 0 . 05 , LFC > 1 ) , significantly increased in the plasma of C57BL / 6J and ADX mice ( C57BL / 6J P ≤ 0 . 05 and ADX P ≤ 0 . 05 , LFC > 1 ) , and significantly increased in the plasma of ADX mice only ( C57BL / 6J P > 0 . 05 and ADX P ≤ 0 . 05 , LFC > 1 ) .

(A) Female C57BL/6J and ADX mice were put in normoxia and hypoxia for 6h and 24h. Plasma was isolated for metabolomics analysis. The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL/6J mice (C57BL/6J P ≤ 0.05 and ADX P > 0.05, LFC > 1), significantly increased in the plasma of C57BL/6J and ADX mice (C57BL/6J P ≤ 0.05 and ADX P ≤ 0.05, LFC > 1), and significantly increased in the plasma of ADX mice only (C57BL/6J P > 0.05 and ADX P ≤ 0.05, LFC > 1).


( D - G ) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL / 6J mice injected with 5 mg RU486 or vehicle ( DMSO ) ( D , F ) , and in GRdim / dim mice and their wild - type littermates ( E , G ) after 6h of normoxia and hypoxia . N = 4 - 9 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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 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

(D-G) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (D, F), and in GRdim/dim mice and their wild-type littermates (E, G) after 6h of normoxia and hypoxia. N=4-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( H - J ) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL / 6J and ADX mice ( H ) , in female C57BL / 6J mice injected with 5 mg RU486 or vehicle ( DMSO ) ( I ) , and in GRdim / dim mice and their wild - type littermates ( J ) . N = 5 - 9 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(H-J) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice and their wild-type littermates (J). N=5-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( A ) Female C57BL / 6J mice ( n = 3 per group ) were put in normoxia or hypoxia for 24h , injected with PBS or DEX ( 10 mg / kg ) and 2h later , indicated organs were isolated and gene expression was measured via RT - qPCR . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(A) Female C57BL/6J mice (n=3 per group) were put in normoxia or hypoxia for 24h, injected with PBS or DEX (10 mg/kg) and 2h later, indicated organs were isolated and gene expression was measured via RT-qPCR. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( B ) LPS LD100 dose - response in female C57BL / 6J mice injected with indicated LPS doses after 24h hypoxia . During the follow - up of lethality , mice remained under hypoxic conditions . N - values are indicated in the legend .

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

(B) LPS LD100 dose-response in female C57BL/6J mice injected with indicated LPS doses after 24h hypoxia. During the follow-up of lethality, mice remained under hypoxic conditions. N-values are indicated in the legend.


( E ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia . Liver was isolated 6h after injected and typical GRE genes were measured via RT - qPCR . N = 4 - 5 per group . ( F ) Female ADX mice were put in normoxia or hypoxia for 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) and 2h later , liver was isolated for RT - qPCR analyses of typical GRE genes . N = 3 - 4 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(E) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia. Liver was isolated 6h after injected and typical GRE genes were measured via RT-qPCR. N=4-5 per group. (F) Female ADX mice were put in normoxia or hypoxia for 24h, injected i.p. with PBS or DEX (10 mg/kg) and 2h later, liver was isolated for RT-qPCR analyses of typical GRE genes. N=3-4 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( G ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia , with or without pre - treatment with 10 mg / kg DEX 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . Mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) .

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

(G) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia, with or without pre-treatment with 10 mg/kg DEX 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. Mice in normoxia: black circles (LPS), black squares (DEX-LPS); mice in hypoxia: white circles (LPS), white squares (DEX-LPS).


( H ) Female ADX mice were injected with 0 . 05 mg / kg LPS after 24h normoxia or hypoxia , , with or without 10 mg / kg DEX pre - treatment 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . ADX mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; ADX mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) .

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

(H) Female ADX mice were injected with 0.05 mg/kg LPS after 24h normoxia or hypoxia, , with or without 10 mg/kg DEX pre-treatment 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. ADX mice in normoxia: black circles (LPS), black squares (DEX-LPS); ADX mice in hypoxia: white circles (LPS), white squares (DEX-LPS).


a ) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3 , 5 , 12 and 24 weeks ( bar represents 50 μm ) . In each image a clone is circled in white , illustrating the progression of clone size at different times . b ) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42 . The frequency of larger clones increases over time . Chi square test p < 0 . 0001 for P21 group and p = 0 . 0056 for P42 group . The number of clones evaluated at each time point is indicated above each histogram series . c ) and d ) Correction for contiguous clones harbouring a same colour .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3, 5, 12 and 24 weeks (bar represents 50 μm). In each image a clone is circled in white, illustrating the progression of clone size at different times. b) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42. The frequency of larger clones increases over time. Chi square test p<0.0001 for P21 group and p=0.0056 for P42 group. The number of clones evaluated at each time point is indicated above each histogram series. c) and d) Correction for contiguous clones harbouring a same colour.