Fig 2: Increased thomsbospondin-1 (TSP1) expression in mice after IH exposure.(A) C57BL/6 mice were housed under normoxia or IH with or without infusion of Ang II for 28 days. Plasma concentration of TSP1 in mice detected by ELISA. (B) Thbs1 mRNA level in left ventricle of mice exposed to IH with or without infusion of Ang II for 14 days quantified by RT-PCR. (C) C57BL/6 mice were housed under normoxia or IH with or without infusion of Ang II for 28 days. The protein levels of TSP1, phosphorylated Smad2/3 (p-Smad2/3), and total Smad2/3 (t-Smad2/3) in left ventricle of mice detected by western blot analysis. (D) Quantification of TSP1 and p-Smad2/3 in (C). (E) Representative confocal microscopy images of immunofluorescence staining for TSP1 and DAPI. Scale bar, 20 µm. (F) Quantification of TSP1 fluorescent intensity in (E). Data are mean ± SEM, n = 6 mice per group, *p<0.05, 2-way ANOVA with Bonferroni post-test. (G) Quanti?cation of number of cells negative for CD45, CD31, CD11b, and Ter119 and positive for Thy1 from left ventricle tissue digestion, that stained positive for intracellular TSP1 by ?ow cytometry. Data are mean ± SEM, n = 6, *p<0.05, unpaired 2-tail t test. (H) Plasma concentration of TSP1 in healthy individuals and patients with obstructive sleep apnea (OSA) detected by ELISA. Data are mean ± SEM, n = 21, *p<0.05, unpaired 2-tail t test. Data used for quantitative analyses as well as the numerical data that are represented in graphs are available in Figure 2—source data 1.Figure 2—source data 1.Intermittent hypoxia (IH) increased thomsbospondin-1 (TSP1) expression.

Fig 3: ZsGreen signals in erythroid progenitor cells in the fetal liver and adult bone marrow. (A) ZsGreen signals in c-kit+ or c-kit− cells from the ZfatZsG/+ (black line) and WT (gray-filled) fetal liver at E12.5. (B) Definition of stages during erythroid maturation in the fetal liver at E12.5. Fetal liver cells from ZfatZsG/+ embryos were stained with CD71 and Ter119, and analyzed by flow cytometry. P1, CD71-Ter119low; P2, CD71+Ter119low; P3, CD71+Ter119mid; and P4, CD71+Ter119high. (C) ZsGreen signals in P1-P4 cells from the ZfatZsG/+ (black line) and WT (gray-filled) fetal liver at E12.5. (D) ZsGreen signals in c-kit+ cells in the P1-P3 population from the ZfatZsG/+ (black line) and WT (gray-filled) fetal liver at E12.5. (E) ZsGreen signals in the bone marrow cells isolated from 4-week-old ZfatZsG/+ (black line) and WT (gray-filled) mice. (F) Definition of stages during erythroid maturation in the adult bone marrow. Bone marrow cells from ZfatZsG/+ mice were stained with CD71 and Ter119, and analyzed by flow cytometry. P1, CD71−Ter119low; P2, CD71+Ter119low; P3, CD71+Ter119mid; P4, CD71+Ter119high; and P5, CD71−Ter119high. (G) ZsGreen signals in P1-P5 cells from the ZfatZsG/+ (black line) and WT (gray-filled) bone marrow. Data are representative of three independent experiments. CD, cluster of differentiation; E, embryonic day; WT, wild-type; Zfat, zinc finger and AT-hook domain containing.

Fig 4: Pleckstrin-2 knockdown in vitro induces cell death and decreases enucleation particularly in fetal liver cells from ß-thalassemic embryos.a Flow cytometry analysis of nucleated (Q2) and enucleated (Q4) cells after nt-shRNA, and nucleated (Q6) and enucleated (Q8) cells after plek2-shRNA transfection in early stage (CFU-E) erythroblasts derived from Ter119-negative fetal liver cells (FLCs) from E13.5 WT and ß-thalassemic embryos, revealing the expected higher degree of proliferation and an enucleation defect in ß-thalassemic relative to WT nt-shRNA transfected cells; plek2-shRNA transfection result in a decreased fraction of nucleated (Q6) and enucleated (Q8) cells in WT and ß-thalassemic cells. b Flow cytometry analysis of (a) demonstrates a more severe effect on enucleation in ß-thalassemic plek2-shRNA transfected cells compared to WT plek2-shRNA transfected cells. c Flow cytometry analysis of cell death using 7-AAD demonstrates no difference between WT and ß-thalassemic nt-shRNA transfected FLCs with significant increase in cell death in plek2-shRNA transfected WT and especially ß-thalassemic FLCs (from (a)). (n = 3 mice/group, experiment repeated twice). All data are reported as mean ± s.e.m. and p < 0.05 (one-way ANOVA) was considered statistically significant. (**p = 0.005 vs. WT nt-shRNA; +p = 0.05 vs. nt-shRNA; ++p = 0.005 vs. nt-shRNA) WT = wild type; plek2 = pleckstrin-2; MFI = mean fluorescence index; GFP = green fluorescent protein.

Fig 5: Overexpression of SOX4 in preadipocytes diminished the pool of preadipocytes under HFD condition3-week-old WT and Pref1-Sox4 mice were housed at room temperature and fed with NCD or HFD for 20 weeks.(A) Pref1-Sox4 mice were generated by directly inserting coding sequence (CDS) sequence of Sox4 after a 6-kbp fragment upstream of the transcriptional start site of the Pref1 promoter.(B) iWAT SVFs were isolated from NCD or HFD-fed WT and Pref1-Sox4 mice and cultured for 2 h to exclude the non-adherent cells. Then, the cultured cells were collected and the expression of Sox4 was detected by qPCR (n = 4–6). mRNA levels were normalized to 18s RNA. The mean CT was converted to relative expression value by the equation 2-?Ct.(C) The expression of SOX4 in iWAT SVFs of HFD-fed WT and Pref1-Sox4 mice was detected by Western blotting.(D) The expression of Sox4 in indicated cell populations sorted by flow cytometry was examined by qPCR (n = 4). mRNA levels were normalized to 18s RNA. The mean CT was converted to relative expression value by the equation 2-?Ct.(E–G) The proportions of CD45-: CD31-: Ter119-: PDGFRa+ (Lin-: PDGFRa+ cells) adipocyte precursors (E), DPP4+ MSCs (F) and ICAM1+ preadipocytes (G) were analyzed by flow cytometry in NCD- and HFD-fed WT and Pref1-Sox4 mice (NCD group: n = 4; HFD: n = 4–5).(H) The proportions of MSCs with adipogenic potential (Lin-: CD34+: CD29+), adipocyte progenitors (Lin-: CD34+: CD29+: Sca-1+: CD24+, CD24+ cells), committed preadipocytes (Lin-: CD34+: CD29+: Sca-1+: CD24-, CD24- cells), and nonadipogenic fibroblasts (Lin-: CD34+: CD29+: Sca-1-, Sca-1- cells) were analyzed by flow cytometry in NCD- and HFD-fed WT and Pref1-Sox4 mice (NCD group: n = 3; HFD: n = 4–6).3-week-old WT and Pref1-Sox4 mice with HFD for 20 weeks were used in this study.Data are represented as mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.