Figure 1 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) 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.(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.


Figure 4 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.(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.


Figure 5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) 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.(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.


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3Female 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Female 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).(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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.


Figure 7 ( 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 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.(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).(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).


Figure 1a ) 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 . d ) Plots show the probability for an individual keratinocyte to belong to a clone of a given size . Experimental data are represented in green bars . Red curve represents the 5 - colour simulation and the blue curve the real size simulation . Although the frequency of large clones is overestimated in experimental data due to the limited 5 - color palette , it is apparent that real clone size progresses over time . e ) Histogram charts showing the distribution of clones at D1 - 3 following tamoxifen induction according to total number of cells and number of basal cells . f ) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining . The frequency of larger clones increases over time . Chi square test p = 0 . 0013 for P21 group and p = 0 . 0296 for P42 group . All data are represented as mean ± s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1a) 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.d) Plots show the probability for an individual keratinocyte to belong to a clone of a given size. Experimental data are represented in green bars. Red curve represents the 5-colour simulation and the blue curve the real size simulation. Although the frequency of large clones is overestimated in experimental data due to the limited 5-color palette, it is apparent that real clone size progresses over time.e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells.f) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.


Figure 2a ) Clones were categorized as attached or not attached to hair follicles . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over time . T - test , * * * p < 0 . 0001 , * * p < 0 . 001 . ( Number of clones : 1 week n = 271 , 3 weeks n = 1847 , 5 weeks n = 517 , 12 weeks n = 706 , 24 weeks n = 258 ) . Data are represented as mean ± s . d . b ) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks . The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF . The orange and pink clones are not attached to the HF . Limits of clones ( lines ) and cells ( dots ) within each clone were identified on this optical section . White dashed circles represent the projection of hair follicle openings ( scale bar = 50 µm ) . c ) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination . In the P42 group , clones attached to HF were more likely to be bigger than those not attached at 3 , 5 , 12 and 24 weeks . ǂǂǂǂ Chi square test p < 0 . 0001 . The number of clones analyzed at each time - point is indicated on the graph . Of note , whether attached or not clones continued to grow over time : * * * * Chi square test p < 0 . 0001 . All data are represented as mean ± s . e . m . d ) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size ( dashed line : predicted curve based on theoretical probabilities according to spacing of hair follicles at different time - points ; squares : experimental data ) . Between different time points the distance between hair follicles varies slightly ; therefore each time point ( 3 , 5 , 12 and 24 weeks ) has a distinct theoretical curve . The observed frequency of clone attachment to hair follicles is at every time point above the random prediction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2a) Clones were categorized as attached or not attached to hair follicles. Average clone size of attached (circles) and non-attached (squares) clones were plotted over time. T-test, *** p<0.0001, ** p<0.001. (Number of clones: 1 week n = 271, 3 weeks n = 1847, 5 weeks n = 517, 12 weeks n = 706, 24 weeks n = 258). Data are represented as mean ± s.d.b) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks. The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF. The orange and pink clones are not attached to the HF. Limits of clones (lines) and cells (dots) within each clone were identified on this optical section. White dashed circles represent the projection of hair follicle openings (scale bar= 50 µm).c) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination. In the P42 group, clones attached to HF were more likely to be bigger than those not attached at 3, 5, 12 and 24 weeks. ǂǂǂǂ Chi square test p<0.0001.The number of clones analyzed at each time-point is indicated on the graph. Of note, whether attached or not clones continued to grow over time: ****Chi square test p<0.0001. All data are represented as mean ± s.e.m.d) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size (dashed line: predicted curve based on theoretical probabilities according to spacing of hair follicles at different time-points; squares: experimental data). Between different time points the distance between hair follicles varies slightly; therefore each time point (3, 5, 12 and 24 weeks) has a distinct theoretical curve. The observed frequency of clone attachment to hair follicles is at every time point above the random prediction.


Figure 3a ) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at the time of tissue collection . Clones connected to hair follicles were more likely to be larger in anagen compared to telogen ( Chi square test , * * * p = 0 . 0009 at 3 weeks , * * * p < 0 . 0001 at 5 weeks , * p = 0 . 0193 at 12 weeks , * * * p = 0 . 0001 at 24 weeks ) . However no difference between the two phases was observed in clones that were not attached to HFs . Clone numbers are indicated on the graph . All data are represented as mean ± s . e . m . b ) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post - induction . Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over the number of club hair . T - test , * * * * p < 0 . 0001 . c ) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at induction . Even at a rate of one cell division per 24h , 7 cycles of divisions results in 128 cells . Therefore the findings reported are not incompatible with our claims as suggested . Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe . Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones . Chi square test , * * * p = 0 . 001 ; * * p = 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3a) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m.b) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001.c) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.


Figure 4 ( a - b ) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway ( a ) and corresponding scatter plots quantification . ( Scale bar 100µm ) ( b ) . Analysis focused on the basal layer of the IFE . Each dot represents one image . Proliferation in the IFE is significantly increased during the anagen phase except in mid - anagen . Unpaired t test , * * * * p < 0 . 0001 , * * p = 0 . 0020 . EA n = 32 , EMA n = 13 , MA n = 15 , C n = 21 , T n = 19 from 6 mice . Black arrows : positive Ki67 cells . ( c ) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti - Ki67 antibody or upon BrdU incorporation . Ki67 + cells are labelled with Alexa 647 ( red ) . The density of Ki67 + cells increased in the anagen phase ( iii and iv ) compared to telogen ( i and ii ) . White arrows : ki67 + cells . ( Scale bar = 20µm ) . Similarly , short term BrdU pulse ( 2h ) resulted in significant staining of anagen epidermal areas ( vi , red nuclear stain ) reinforcing hair follicle openings as compared to telogen areas .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(a-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells.(c) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.


Figure 5a - b ) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . a ) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti - BrdU antibody ( red ) . White dots represent hair follicles at different chase time points . a - b ) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . b ) At 5 weeks of chase , BrdU staining ( green ) in skinbowskin revealed single cells distant from hair follicles . White dashed circles : hair follicles . White line : small clones . White arrows : BrdU + cells . ( scale bar = 20 µm ) . c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . c ) Photomicrographs show optical sections from a confocal image stack acquired on a 0 . 5 mm back skin section immunostained with anti - ki67 antibody . Ki67 + cells are labelled with Alexa 647 ( red ) . Back skin from treated and untreated areas was collected 48hrs after TPA topical application . c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . d ) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti - BrdU ( green ) . Small clones not attached to HF ( 2 examples figured by dashed lines ) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation . e ) ( i to iv ) Representative individual sections from Z stacks , show small clones ( lines ) identified in back skin treated ( i and ii ) or not with TPA ( iii and iv ) . ( scale bar = 100 µm ) . ( v to viii ) Maximal intensity projection from stack acquired on whole mounted skin . A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA ( v to vi ) compared to skin treated with vehicle ( vii and viii ) . ( scale bars = 100 µm ) . f ) Reduction in small clone frequency in the areas treated with TPA . Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse . Mann Whitney test , * * p = 0 . 0079 . Data pooled from 3 mice represented as median with interquartile range . The number of clones analyzed is indicated on the graph . * p = 0 . 0272 . Data are represented as mean ± s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points.a-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm).c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.


Figure 6a ) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42 . Data are represented as mean ± s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6a) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42. Data are represented as mean ± s.e.m.


Figure 1 ( G ) Sorted human monocytes were incubated with control medium or SARS - CoV - 2 viruses at an MOI of 1 for 1 h , followed by washing away extracellular viruses and further culture for 1 h . Cells were fixed and stained with the indicated antibodies . Otherwise , monocytes were challenged with 1 μg / ml LPS for 3 h and 10 μM nigericin for 30 min , followed by fixing and staining with the indicated antibodies . Treatment with medium from uninfected Vero cells served as a control . Scale bar : 5 μm . Data information : results were repeated for three times with similar results . Supernatants from control or SARS - CoV - 2 infected CD14 + monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL - 1β levels ( H ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results . Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells ( I ) . The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants ( J ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . results were repeated for three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(G) Sorted human monocytes were incubated with control medium or SARS-CoV-2 viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS for 3 h and 10 μM nigericin for 30 min, followed by fixing and staining with the indicated antibodies. Treatment with medium from uninfected Vero cells served as a control. Scale bar: 5 μm. Data information: results were repeated for three times with similar results.Supernatants from control or SARS-CoV-2 infected CD14+ monocytes with or without LPS and nigericin stimulation were collected and subjected to an ELISA assay for IL-1β levels (H). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: , Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants (J). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.


Figure 2 ( B , C ) THP - 1 cells stably expressing the indicated SARS - CoV - 2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were collected for an ELISA assay to determine supernatant IL - 1β levels ( B ) and cell pellets were subjected to a cell viability assay to verify the percentage of viable cells through checking cellular ATP levels ( C ) . For B - C , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . NS , non - significant . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times . ( D - F ) THP - 1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants and cell pellets were collected and immunoblotted with antibodies against the indicated proteins ( D ) . The percentage of viable cells was determined through checking cellular ATP levels in cell pellets ( E ) . The percentage of cells undergoing pyroptosis was calculated through checking released LDH in the supernatants ( F ) . For E , F , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Similar results were observed for at least three times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 3 ( D ) GSDMD + / + and GSDMD - / - THP - 1 cells were transfected with plasmids encoding nucleocapsid for 24 h , followed by cell fixation , permeabilization and stain with antibodies against nucleocapsid and GSDMD . The nucleus was counterstained with DAPI . Scale bar : 5 μm . ( E , F ) THP - 1 ( E ) or human CD14 + monocytes ( F ) were transfected with plasmids encoding FLAG - tagged nucleocapsid for 36 h , followed by pretreatment of 1 μg / ml LPS for 3 h and 10 μM nigericin stimulation for 30 min . Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG . Input and precipitates were immunoblotted as indicated . ( I ) GST - GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG - tagged full - length nucleocapsid or truncated nucleocapsid lacking amino acids 70 - 160 and 290 - 360 , followed by a GST - pulldown assay . Input and precipitates were immunoblotted as indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) GSDMD+/+ and GSDMD-/- THP-1 cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain with antibodies against nucleocapsid and GSDMD. The nucleus was counterstained with DAPI. Scale bar: 5 μm.(E, F) THP-1 (E) or human CD14+ monocytes (F) were transfected with plasmids encoding FLAG-tagged nucleocapsid for 36 h, followed by pretreatment of 1 μg/ml LPS for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.(I) GST-GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG-tagged full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted as indicated.


Figure 4 ( A , B ) THP - 1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were either immunoblotted with antibodies against the indicated proteins ( A ) or stained with propidium iodide ( PI ) ( B , left ) . Scale bar : 20 μm . The percentage of PI positive cells was calculated ( B , right ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times . ( H , I ) THP - 1 cells stably expressing full - length nucleocapsid or mutant lacking amino acids 70 - 160 and 290 - 360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were either immunoblotted with antibodies against the indicated proteins ( H ) . Cells were also stained with PI and the percentage of PI positive cells was calculated ( I ) . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; NS , non - significant . Results were repeated for three times with similar results . Data information : control vector used here was empty vector corresponded to the nucleocapsid - containing vector . Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Similar results were observed for at least three times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.(H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.


Figure 5 ( A ) GST or GST - GSDMD , p20 ( C285A ) - His , p10 - His and increasing amount of His - nucleocapsid were incubated for a GST - pulldown assay , followed by immunoblotting with antibodies against the indicated protein tags . Data information : Experiments were repeated for three times with similar results . ( G , H ) Wild - type GSDMD and GSDMDsusti . ( G ) or mCherry - GSDMD - C ( H ) were purified from HEK293T cells and incubated with recombinant p20 / p10 complex and His - tagged nucleocapsid in a cleavage buffer , followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C - termimus . Data information : Experiments were repeated for three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) GST or GST-GSDMD, p20 (C285A)-His, p10-His and increasing amount of His-nucleocapsid were incubated for a GST-pulldown assay, followed by immunoblotting with antibodies against the indicated protein tags. Data information: Experiments were repeated for three times with similar results.(G, H) Wild-type GSDMD and GSDMDsusti. (G) or mCherry-GSDMD-C (H) were purified from HEK293T cells and incubated with recombinant p20/p10 complex and His-tagged nucleocapsid in a cleavage buffer, followed by immunoblotting with antibodies against the indicated protein tags or antibody against GSDMD C-termimus. Data information: Experiments were repeated for three times with similar results.


Figure 6GSDMD - / - THP - 1 cells rescued with wild - type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Cells were immunoblotted with antibodies against the indicated proteins ( A ) ( E , F ) GSDMD - / - THP - 1 cells rescued with wild - type or linker substituted GSDMD were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular viruses and further culture for 3 h . Otherwise , cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h . Supernatants were subjected to an ELISA assay to determine IL - 1β levels ( E ) . Otherwise , cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells ( F ) . For E , F , Student ' s t - test was used and data were shown as means ± SD of three technical replicates . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; NS , non - significant . Results were repeated for three times with similar results . ( G ) Sorted CD14 + monocytes from donors were infected with SARS - CoV - 2 at an MOI of 1 for 1h , followed by washing away extracellular viruses and further culture for 3 h . Cells were immunoblotted with antibodies against the indicated proteins . CD14 + monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg / ml LPS for 3 h and served as a positive control . Data information : experiments were repeated for three time with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (A)(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.(G) Sorted CD14+ monocytes from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control. Data information: experiments were repeated for three time with similar results.