EMBO/SourceData huggingface dataset:
Figure 2SARSCoV-2 MA30 infection in C57BL/6 mice 7 days follow up. PEGSerp-1 treatment significantly improved Mean (C) weights (N = 20 mice, C mean ± SD)) Data analysis information: Mean ± SD ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Unpaired T tests; indicated by line at top with subgroup analyses in brackets below; Prophylactic treatment black - Saline, red -PEGSerp-1 treatments.E Micrographs illustrate lung consolidation in Saline and PEGSerp-1 treated lungs. Large blue arrows indicate areas of consolidation in H&E stained lung sections (Mag 2X).G, Combined weights for the 4 and 7 day follow up studies demonstrate consistently improved mean weights (G; p < 0.0153- 0.0001) with PEGSerp-1 treatments in MA30 infected mice. (N = 39mice, mean ±SD) Data analysis information: black - Saline, red -PEGSerp-1 treatments.I Micrographs illustrate lung consolidation divided by total section analyzed in Saline and PEGSerp-1 treated MA30 infected mice at 4 days follow up.PEGSerp-1 delayed treatment at 10ng/gm starting 2 days after infection reduced weight loss (L; p < 0.001-0.05). (N = 24 mice, mean ± SE) Data analysis information: Mean ± SE ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; blue - Saline, green and blue - PEGSerp-1.
Figure 3A, B Micrographs illustrating iNOS positive M1 macrophage cell infiltrates on IHC stained lung sections in Saline and PEGSerp-1 (A) treated mice with significantly reduced IHC positive cell counts at 4 days follow up after PEGSerp-1 prophylactic treatment (B; *p < 0.045). IHC stained lung sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.C, D iNOS positive infiltrates in IHC stained lung sections with Saline or PEGSerp-1 (C) treatment with significantly reduced IHC positive cell counts at 7 days follow up with prophylactic PEGSerp-1 treatment (D; **p < 0.0052). IHC stained lung sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.E Delayed PEGSerp-1 10ng/gm dose treatment given 2 days after infection also reduced iNOS positive cell counts (*p < 0.0368). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; blue circles Saline, red and green circles PEGSerp-1 treatments.G CD3 + T cell counts were not significantly altered at 4 days (p = 0.1058). H CD4 + T cell counts are not reduced at 4 days (p =0.5031) but are reduced at 7 days (*p < 0.0454) with PEGSerp-1 treatments. I CD8 positive T cell counts on IHC stained sections have a trend toward an increase at 4 days (p = 0.1503) and a significant reduction at 7 days (*p < 0.0303) with PEG Serp-1 treatments. J IHC for Neutrophil marker Ly6G did not detect significant changes in PEGSerp-1 treatment at days 4 (p = 0.0860) and 7 (p = 0.6570). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments.
Figure 4iNOS M1 stained macrophage invasion in Saline control treated mice (A) Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).PEGSerp-1 treatment reduced iNOS + macrophage infiltration. iNOS M1 stained macrophage invasion is significantly reduced with PEGSerp-1 treatment (B). Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).H & E stained histology sections of lung illustrating consolidation in Saline treated mice (E) saline treated uninfected mouse tissues had similar levels of consolidation Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).PEGSerp-1 treatment reduced lung consolidation H & E stained histology sections of lung illustrating consolidation is reduced by PEGSerp-1 treatment (F). Black arrows point to brown iNOS positive macrophage on IHC stained lung sections (Mag 40X).
Figure 5A, B Micrographs illustrating iNOS positive cells in Saline (A) and PEGSerp-1 (B) treated mice at 4 days follow up. IHC stained sections 40X.
Figure 6PEGSerp-1 treated mouse lung sections from MA10 at day 2 follow up have significant reduction in uPAR positive stained areas (A) with reduced positively stained hemorrhagic areas in lungs at 2 days in MA10 infected BALB/c (B; p < 0.041) and reduced uPAR + stained cell counts at 4 days (C; p < 0.0202) and 7 days (C in alveoli (p < 0.0148) in MA30 infected C57BL/6 mice. With delayed treatments uPAR is reduced at 7 days follow up (D; ANOVA p< 0.0001, p < 0.0002 for PEGSerp-1 at 10µg/kg). FX staining was reduced with prophylactic PEGSerp-1 at day 7 but not at day 4 follow up (E; ANOVA p < 0.0008, p < 0.0001 for day 7 and p = 0.5148 for day 4). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatmentsC5b-9 staining is reduced in prophylactic PEGSerp-1 treatment in MA30 infected C57BL/6 mice on IHC stained sections (H) with significant reductions at day 4 (I; p <0.0114), and a nonsignificant trend at day 7 follow up (p = 0.5694). With delayed PEGSerp-1 treatment C5b/9 detection on IHC is significantly reduced by both PEGSerp-1 doses (J; p < 0.0131). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments
Figure 7D-G In MA30 infected C57BL/6 mice increased S protein is seen on IHC staining seen in lung sections from Saline treated mice (D) is reduced with PEGSerp-1 treatment at 7 days follow up (E, F; p < 0.001) and in the small vessels (G; p < 0.0364). Data information: Mean ±SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below.
Figure 8A, B Relative gene expression is significantly increased for SARS-CoV-2 MA30 viral E gene (A; p < 0.0017) and interferon response ISG15 (B; p < 0.0044) in Saline treated infected lung extracts at 4 days follow up, when compared to uninfected mouse lungs. Data information: Gene expression is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in inflammatory and coagulation responses are increased after SARS-CoV-2 MA30 infection in lung and heart tissues (SARSCoV-2 MA30 infection - N = 18 mice 4 days follow up; N = 20 mice 7 days follow up; Uninfected mice - N = 2). PEGSerp-1 treatment significantly modified gene expression for uPAR and complement pathways. C-L Inflammatory cytokines and growth factors are significantly increased with infection; IL-1 (C; p < 0.0003), IL-6 (D p < 0.0001), IL-10 (E; p < 0.0001) ), TNF (F; p < 0.0044), TGFb (G; p < 0.0022) are increased with SARS-CoV-2 infection at 4 days follow up; with VEGF the exception (H; p = 0.4695 ) Factors in the coagulation pathway, fX (I; p < 0.0001), tPA (J; p < 0.0382), and neuroserpin (K; p = 0.1883 ) are also increased. None of these markers are modified by PEGSerp-1 treatments. Data information: Gene expression is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).
Figure 1C, Heatmap of DEGs between the Src-1/-2 dKO and WT fetuses that are involved in the lung development pathway (GO: 0030324).
Figure 2A and C, mRNA levels of Arg1 and Ass1 in fetal lungs of mice with different genotypes at 18.5 dpc; n=8 biological replicates in each group for panel A and 7 biological replicates in each group for panel C. One-way ANOVA followed by Tukey's multiple comparison test was used to analyze the data. The data are shown as the mean ± s.e.m., *P<0.05, ***P<0.001, ****P<0.0001, compared with the WT group.B and D, Representative immunoblots of ARG1 and ASS1 protein expression in fetal lungs from mice with different genotypes at 18.5 dpc. Densitometric quantitation of immunoblot scans (relative to β-actin as a loading control) is also shown as a bar graph; n=3 biological replicates in each group. One-way ANOVA followed by Tukey's multiple comparison test was used to analyze the data. The data are shown as the mean ± s.e.m., *P<0.05, **P<0.01, ***P<0.001, compared with the WT group.E and F, Immunohistochemical staining of ARG1 and ASS1 in fetal lungs of WT and Src-1/-2 dKO mice at 18.5 dpc. PBS was used instead of the primary antibody as a negative control (NC). Scale bars: 50 μm in E and F.
Figure 3A and C, mRNA expression of Arg1 and Ass1 in the lungs of WT fetuses at gestational ages from 15.5 dpc to labor. n=6 biological replicates for each gestational age in panel A; n=6 biological replicates for 15.5dpc, 17.5dpc, and labor group, n=5 biological replicates for 16.5dpc and 18.5dpc group in panel C. Kruskal-Wallis test followed by Dunn's multiple comparison test was used to analyze the data. The data shown are the mean ± s.e.m. **P<0.01, ****P<0.0001.B and D, Representative immunoblots of gestational changes in ARG1 and ASS1 protein expression in the lungs of WT fetuses at gestational ages from 15.5 dpc to labor. Densitometric quantitation of scans of immunoblots (relative to tubulin as loading control) are also shown as the mean ± s.e.m. in bar graph; n=4 biological replicates for each gestational age; Kruskal-Wallis test followed by Dunn's multiple comparison test was used to analyze the data. *P<0.05 compared with 15.5 dpc.E and F, Immunohistochemical staining of ARG1+ (E) or ASS1+ (F) cells in the fetal lungs of WT mice at 15.5 dpc, 17.5 dpc and labor. PBS was used instead of the primary antibody in the NC group. Scale bars: 100 μm in E and F.
Figure 4D and E, Histograms showing the content of arginine, ornithine, ADMA and the ratio of arginine to ADMA in the fetal lungs of WT mice and Src-1/-2 dKO mice at 18.5 dpc. Mann Whitney test was used to analyze the data in panel D, and unpaired t test was used to analyze the data in panel E. n=5 biological replicates for each group, and the data are shown as the mean ± s.e.m. The central band indicates the median, the boxes indicate the upper quartile and lower quartile, and the whiskers indicate maximum value and minimum value of the data. *P<0.05, **P<0.01, compared with the WT fetal lungs.
Figure 5B, Representative immunoblots of ARG1 expression in the fetal lungs from the AAV-shArg1-injected group and the AAV-2/9-injected group. Densitometric quantitation of scans of immunoblots (normalized to tubulin) is also shown as a bar graph. Unpaired t test was used to analyze the data and n=3 biological replicates for each group. The data are shown as the mean ± s.e.m. **P<0.01 compared with the AAV-2/9-injected group.C, Representative immunoblots and densitometric quantitation of Ki67, caspase3 precursor (pro-caspase3) and cleaved caspase3 expression in the fetal lungs from the AAV-shArg1-injected group and AAV-2/9-injected group. Unpaired t test was used to analyze the data and n=3 biological replicates for each group. The data are shown as the mean ± s.e.m. **P<0.01 compared with the AAV-2/9-injected group; n.s indicates non-significance.E, Multiple immunofluorescence staining and densitometric quantitation of TUNEL with PDPN antibody (type I alveolar epithelial cells marker) on sections of fetal lungs from AAV-shArg1-injected mice vs. AAV-2/9-injected mice. Blue: DAPI (nuclei); Green: PDPN (type I cells); Red: TUNEL positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. F, Multiple immunofluorescence staining and densitometric quantitation of TUNEL with LPCAT1 antibody (type II alveolar epithelial cells marker) on sections of fetal lungs from AAV-shArg1-injected mice vs. AAV-2/9-injected mice. Blue: DAPI (nuclei); Green: LPCAT1 (type II cells); Red: TUNEL positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. Data information: Scale bars in E and F: 50 μm.
Figure 6B, Quantitative analysis of the gel area containing hTERT-HM cells treated with L-arginine or vehicle control. Mann Whitney test was used to analyze the data, and n=9 biological replicates in control group, n=12 biological replicates in L-Arginine-treated group. The data are shown as the mean ± s.e.m. **P < 0.01. Representative images of spontaneous contractions of the gel disks are shown.C, Representative western blots and quantitative analysis of CAP expression in the myometrium of pregnant mice injected intraplacentally with AAV-shArg1 or AAV control viruses. Mann Whitney test was used to analyze CX43 and PTGFR data and unpaired t test was used to analyze COX2 and OXTR data. n=4 biological replicates for CX43, COX2 and PTGFR groups, and n=5 biological replicates for OXTR group. The data are shown as the mean ± s.e.m. *P < 0.05.D, Representative western blots and quantitative analysis of NF-κB p-p65 (phospho S536) and NF-κB p65 expression in the myometrium of pregnant mice injected intraplacentally with AAV-shArg1 or AAV control viruses. Unpaired t test was used and n=4 biological replicates for each group. The data are shown as the mean ± s.e.m. **P < 0.01.
Figure 7Chromatin immunoprecipitation (ChIP) was performed with WT or Src-1/-2 dKO fetal lung tissues using antibodies against GR (B) Enrichment of these transcription factors at the GRE or C/EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR. Unpaired t test was used for panel B and n=6 biological replicates for each group. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.Chromatin immunoprecipitation (ChIP) was performed with WT or Src-1/-2 dKO fetal lung tissues using antibodies against SRC-1 (C), SRC-2 (D), or C/EBPβ (E). Enrichment of these transcription factors at the GRE or C/EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR. Mann Whitney test was used for Panel C-E. n=6 biological replicates for each group. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. F, Quantitative PCR results for GRE and C/EBP binding site using "input" (positive control) DNA without being immunoprecipitated with any specific antibody. n=6 biological replicates for each group. The data are presented as the mean ± s.e.m.
Figure 1A. Survival curves for WT and Spns2-/- CLP models without antibiotic therapy within 72 hours (n=13 for each group). Data information: Data are presented as percentage (A N represents biological replicates. P values were determined by log-rank test (A *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.B. CFU counting in livers and spleens of CLP models without antibiotic therapy (n=8 per time point for each group). Data information: Data are presented as mean ± s.e.m. (B, N represents biological replicates. P values were determined by unpaired t-test (B, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Concentrations of serum cytokines in CLP models without antibiotic therapy (n=6 per time point for each group). Data information: Data are presented as mean ± s.e.m. C, N represents biological replicates. P values were determined by unpaired t-test C, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.E. Survival curves for WT (n=12) and Spns2-/- (n=17) CLP models receiving meropenem treatment within 7 days. Data information: Data are presented as percentage E) N represents biological replicates. P values were determined by log-rank test E) *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Concentrations of serum cytokines in meropenem-treated CLP models (n=6 per time point for each group). Data information: Data are presented as mean ± s.e.m. G). N represents biological replicates. P values were determined by unpaired t-test G). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
Figure 2A. Gene expression of inflammatory cytokines in isolated PMs challenged with 10 ng/ml LPS (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. (A, N represents biological replicates. P values were determined by unpaired t-test (A, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.B. Survival curves for bone marrow transplanted CLP models without antibiotic therapy within 7 days (n=8 for each group). Data information: Data are presented as percentage (B, N represents biological replicates. P values were determined by log-rank test (B, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Concentrations of serum cytokines in bone marrow transplanted CLP models without antibiotic therapy (n=6 to 8 per time point for each group). Data information: Data are presented as mean ± s.e.m. C, N represents biological replicates. P values were determined by unpaired t-test C, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.D. Survival curves for bone marrow transplanted CLP models receiving meropenem treatment within 7 days (n=8 for each group). Data information: Data are presented as percentage D, N represents biological replicates. P values were determined by log-rank test D, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Survival curves for bone marrow transplanted sepsis models induced by E. coli peritoneal infection within 7 days (n=4 for each group). Data information: Data are presented as percentage G) N represents biological replicates. P values were determined by log-rank test G) *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
Figure 3B. Oxygen consumption rates (OCR) and quantifications of basal respiration, maximal respiration, ATP production, and proton leakage in resting WT, Spns2-/-, and 1 μM S1P pre-treated Spns2-/- PMs (n=4 for each group). Data information: Data are presented as mean ± s.e.m. (B N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.C. Total glucose consumption in 72 hours (n=4 to 5 for each group), the ratio of NAD+ to NADH (n=4 for each group), and the levels of intracellular lactate (n=4 to 5 for each group) in resting PMs. Data information: Data are presented as mean ± s.e.m. C). N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
Figure 4A. Fragmented mitochondrial morphology in Spns2-/- PMs revealed by ransmission electron microscopy. Zoom-in view of the area in the red frame are shown below. Scale bars: 1 μm. B. The lengths of 38 and 47 mitochondria from WT and Spns2-/- PMs, respectively (n=1 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.F. The expression of proteins involved in mitochondrial dynamics (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.G. Heat map displaying the gene expression of MAS and G3PS components (n=3 for each group).H. Protein levels of Slc25a12 and Slc25a13 in WT, Spns2-/-, and 1 μM S1P pre-treated Spns2-/- PMs (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
Figure 5D. Activities of total superoxide dismutase (SOD) and catalase in resting PMs (n=4 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; ***P < 0.001; n.s., not significant.E. Protein levels of peroxiredoxin 1 (Prdx1) and Prdx6 in resting PMs (n=3 per group). Data information: Data are presented as mean ± s.e.m. represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; ***P < 0.001; n.s., not significant.
Figure 6A. Elevated levels of intracellular lactate in resting WT PMs treated with 5 and 10 nM rotenone for 16 hours (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.C. Enhanced activities of total superoxide dismutase (SOD) and catalase in rotenone-treated WT PMs (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.F. Attenuated activities of total SOD and catalase in oxamate- and S1P-treated Spns2-/- PMs (n=3 to 4 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 7A, B. Reduced gene expression of inflammatory cytokines in Spns2-/- PMs treated with either oxamate (A) or mitoquinone (B) (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.C. Activation of Spns2/S1P signaling and suppression of the lactate-ROS axis promoted the survival of Spns2-/- sepsis models induced by heat-killed E. coli through the alleviation of hyerinflammation (n=5 for Spns2-/- control group; n=4 for each treatment group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.
Figure 8A. S1P treatment restored inflammatory response in Spns2-/- PMs after 6 hours post- LPS challenge (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. (A N represents biological replicates. P values were determined by unpaired t-test (A *P < 0.05; **P < 0.01; ***P < 0.001.B. Enhancement of Spns2/S1P signaling significantly improved the survival of CLP models without antibiotic therapy (n=8 for Spns2-/- saline group; n=14 for Spns2+/- group; n=12 for Spns2-/- S1P treatment group). Data information: Data are presented as percentage (B). N represents biological replicates. P values were determined by log-rank test (B). *P < 0.05; **P < 0.01; ***P < 0.001.C. Enhancement of Spns2/S1P signaling restored inflammatory response in Spns2-/- CLP models at 12 hours post-infection (n=6 for each group). Data information: Data are presented as mean ± s.e.m. C) N represents biological replicates. P values were determined by unpaired t-test C) *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 1(C) End fusion analysis for domain deletion mutants of Rap1. NotI-digested genomic DNA from nitrogen-starved cells was analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blot with probes specific for the terminal C, I, L, and M fragments from chromosomes I and II.(D) PFGE analysis as in (C) for nitrogen-starved cells harboring N-terminal truncation mutants of Rap1. All mutants were V5-tagged at their C-terminus.