EMBO/SourceData huggingface dataset:
Figure 2(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFP-NMHCIIA . LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites.(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (B) simultaneously GFP-NMHCIIA and mcherry-KDEL. LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs(C) Confocal microscopy images of HeLa cells left untreated (Control) or treated with LLO (0.5 nM 15 min). Cells were stained with FITC-WGA (Plasma membrane, PM-red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body.(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).(G) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red).(H) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA.(I) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).
Figure 3(A) Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL-proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar - 10 μm. (B-D) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) with wt Lm. Values are the mean ±SEM (n≥3), p-values were calculated using (B-C) one-way-ANOVA with Dunnett post hoc analyses and (D) two-tailed un-paired Student's t-test, *p<0.5, ***p<0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C).(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.(H) Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n≥3), p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01, ***p<0.001. See also Appendix Fig S2 and Fig EV3.
Figure 4(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant(B) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO).(C) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time.(D) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells.(E-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK.(I-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).
Figure 5(A) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.
Figure 6(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.(D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).
Figure 7(A) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates (WCL) and IP fractions (IP Gp96) of extracts of zebrafish larvae 3 dpf uninfected (U) or infected with wt or ∆hly Lm (low dose 200-1500 CFU) for 24h.(B) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control (Ctrl-mo) or Gp96 morpholino oligonucleotides (Gp96 mo).(C) Confocal microscopy images of zebrafish larvae (Ctrl mo or Gp96 mo) infected (low dose) in the tail muscle, with the GFP-expressing wt or Δhly Lm for 24 h, immunolabelled for KDEL-proteins (green), and stained with phalloidin (actin, red) and DAPI (white). Images show actin rich structures at the cell cortex (arrows) and ER-KDEL puncta (arrowheads) in larvae infected with wt strain.(D) CFU counts per zebrafish larvae (Ctrl or Gp96 mo) infected with wt (low dose) or Δhly (high dose > 10,000 CFU) Lm and analysed at 0, 24, 48 hpi. Results are mean ± SEM (n≥3) (horizontal bars) and each circle represents 1 larvae. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01.(E) Survival curves of zebrafish larvae (Ctrl-mo or Gp96-mo) infected with a wt (low dose) or Δhly (high dose > 10,000 CFU) Lm. Results are the mean ± SEM (n≥3). wt infection of Ctrl and Gp96 mo, n=28 larvae; Δhly infection of Gp96 mo n=13. p-values were calculated using Log Rank test, **p<0.01.(F) CFU counts per zebrafish larvae infected with a high dose of ∆hly Lm at different times post-infection. Results are mean ± SEM (n≥3) (horizontal bars); each point represents 1 larvae. See also Fig EV5.
Figure 1(F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.
Figure 2(D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.
Figure 3(E) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.
Figure 4(E) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom.
Figure 5(H-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.
Figure 7(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.
Figure 8(C) ChIP-qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.
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.
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.
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.
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).