Fig 2: IH induced STAT3 signaling activation in CFs.(A) mPCFs were cultured and exposed to IH for the indicated time. The protein levels of phosphorylated JAK1 at Tyr1034/1035 (p-JAK1Y1034/1035), total JAK1 (t-JAK1), p-JAK2 at Tyr1008 (p-JAK2Y1008), total JAK2 (t-JAK2), p-JAK3 at Tyr980/981 (p-JAK3Y980/981), total JAK3 (t-JAK3), p-STAT3 at Tyr705 (p-STAT3Y705) and Ser727 (p-STAT3S727) and total STAT3 (t-STAT3) detected by western blot analysis. (B) Quantification of indicated protein levels in (A). Data are mean ± SEM, n = 4 independent experiments, *p<0.05, 1-way ANOVA with Bonferroni post-test. (C–D) mPCFs were cultured and exposed to IH for 1 hr with or without TG101209 (2 μM). Indicated protein levels were analyzed (D). (E) mPCFs were exposed to IH for 1 hr. Representative confocal microscopy images of immunofluorescence staining for STAT3 and DAPI. Scale bar, 20 μm. (F) Quantification of subcellular localization of STAT3 in (E). Data are mean ± SEM, n = 5 independent experiments, *p<0.05, unpaired 2-tail t test. (G) The protein levels of p-STAT3Y705 and p-STAT3S727 in ventricles of mice with or without IH detected by western blot analysis. (H) Quantification of p-STAT3Y705, p-STAT3S727 and t-STAT3 in (E). Data are mean ± SEM, n = 6 mice per group, *p<0.05, unpaired 2-tail t test. (I) Representative confocal microscopy images of immunofluorescence staining for STAT3, periostin, and DAPI in mice exposed IH with or without Ang II infusion for 4 weeks. The white arrows indicate periostin-positive cells with nuclear localization of STAT3. Scale bar, 10 μm. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 4—source data 1.Figure 4—source data 1.Intermittent hypoxia induced STAT3 signaling activation.

Fig 3: Inhibitory effect of HJ901 on NF-κB and JAK2-STAT3 pathways in vitro. (A–D) All cells lines were treated with or without HJ901 (15 or 30 μM) for 24 h and cell lysates were prepared for analysis of P-NF-κB/NF-κB, P-IκBα/IκBα, P-JAK2/JAK2, and P-STAT3/STAT3 by western blotting. The effect of HJ901 on the expression of P-NF-κB/NF-κB, P-IκBα/IκBα, P-JAK2/JAK2, and P-STAT3/STAT3 proteins are shown for these cell lines. Quantification of relative protein expression was performed by densitometric analysis using β-actin as an internal control. Similar results were obtained from three independent experiments. All data are presented as the means ± SEM (n = 5 in each group). *p < 0.05 and **p < 0.01 vs. PBS group.

Fig 4: A cell non-autonomous action of KSR2 is required for GH signaling in the liver.(a) Hepatic STAT5b and JAK2 phosphorylation in PN17 WT and ksr2−/− mice 15 min after IP injection of PBS or 125 μg/kg recombinant human GH. Liver tissue was lysed and analyzed via western blot with the indicated antibodies. The ratio of phosphorylation to total STAT5 and JAK2 was analyzed by LI-COR Odyssey system (n = 4–5 per group). Quantification is shown to the right of the western blot. (b) STAT5 target genes ALS, MUP3, and MUP1/2/6/8 were measured in livers from PN17 WT and ksr2−/− mice (n = 4–6 per group). WT levels were set to 1. *p < 0.05, **p < 0.01. (c) qPCR of SOCS1-3 and CIS mRNAs from WT and ksr2−/− liver (n = 6–8 per group). For comparison, levels of WT were set to 1 and rps18 was used as an internal control. (d) Muscle STAT5b phosphorylation in PN17 WT and ksr2−/− mice treated as in panel a. (e) IGF-1 mRNA levels from kidney, heart, and muscle of PN17 WT and ksr2−/− mice (n = 3–4 per group). IGF-1 levels of WT were set to 1, and 18srRNA was used as an internal control. (f) STAT5 phosphorylation in primary hepatocytes from PN17 WT and ksr2−/− liver treated for 5 min with 100 ng/ml GH. Results are representative of three independent experiments. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Fig 5: IFNγ production by NK cells is dependent on TYK2 kinase activity. (A) DX5+ cells were MACS-purified from spleens of WT, Tyk2K923E and Tyk2−/− mice, expanded for 7 d with IL-2, stimulated with IL-12 for indicated time points or left untreated (w/o) and further analyzed for activation of STATs and JAK2 and for IFNγ production. Levels of pSTAT3, pSTAT4 and pJAK2 were assessed by Western blot and a representative blot out of two independent experiments is shown (left panel). The production of IFNγ by NK cells was assessed by ELISA (right panel) and mean ± SEM is presented from three independent experiments (n = 3 per genotype per treatment). (B) WT, Tyk2K923E and Tyk2−/− splenocytes were stimulated with anti-NK1.1 antibody for 5 h. Intracellular IFNγ levels were assessed by flow cytometry and the percentage of IFNγ+ cells within the CD3ϵ−DX5+ population (left panel) or level of IFNγ expression (MFI, right panel) is shown as boxplots with whiskers from minimum to maximum from three independent experiments (n = 6−8 per genotype). (C) WT, Tyk2K923E and Tyk2−/− splenocytes were stimulated with anti-NK1.1 antibody for 8 h. Intracellular CCL3 levels were assessed by flow cytometry and the percentage of CCL3+ cells within the CD3ϵ−DX5+ population (left panel) or level of CCL3 expression (MFI, right panel) is shown as boxplots with whiskers from minimum to maximum from two independent experiments (n = 4 per genotype). (A, B, C) *P < 0.05, **P < 0.01, ***P < 0.001.