Fig 2: HDAC inhibition increases HIF-2a expression in UPS.(a) Epas1 mRNA expression of tumours isolated from KP mice 7 weeks after Ad-Cre injection (early; n=4), 8.5 weeks after Ad-Cre injection (late; n=4), and whole-mouse gastrocnemius muscle from uninjected muscle (n=3). (b) EPAS1 mRNA expression of sarcoma cell lines classified as SAHA resistant (9 cell lines), intermediate resistant (Interm. Resistant; 14 cell lines) and sensitive (12 cell lines) from the Oncomine analysis of Garnett et al. data set45. Values are normalized to median-centered intensity, and shown on a log2 scale. *P<0.05, **P<0.01. (c) HIF1A mRNA expression from the Garnett et al. data set (error bars are±s.e.m.). SAHA resistant, 9 cell lines; Intermediate resistant (Interm. Resistant), 13 cell lines; Sensitive, 12 cell lines. (d) Epas1 mRNA expression of KP230 and KP250 mouse UPS cell lines treated with DMSO or SAHA at the indicated drug concentrations. Each bar represents three independent experiments performed in triplicate. *P<0.05, **P<0.01. (e) Hif1a mRNA expression of KP230 and KP250 cells treated with DMSO or SAHA (2 µM). Each bar represents three independent experiments performed in triplicate. (f) Immunoblot for HIF-1a and HIF-2a protein in KP230 and KP250 cells treated with DMSO or SAHA (2 µM), grown under 21% O2 or 0.5% O2 conditions. (g) Serpine1 mRNA expression of KP230 and KP250 cells treated with DMSO or SAHA at the indicated drug concentrations. Each bar represents three independent experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001. (h) ChIP of KP250 cell treated with DMSO or SAHA (2 µM), grown under 21% O2 (N) or 0.5% O2 (H) conditions. ChIP primers used amplified the 500-bp fragment upstream of the Epas1 and Hif1a TSSs. Histone H3 and acetylated histone H3 specific (H3Ac) antibodies were used for ChIP, with IgG as negative control. Input was diluted 1:100. Results are representative of two independent experiments. All data shown are the mean±s.e.m. All P-values were calculated using a two-tailed Student's t-test.

Fig 3: Hif2a deficiency ameliorates renal fibrosis induced by UUO surgery.a Masson-trichrome staining showing fibrosis was reduced. Scale bar = 20 µm. b Quantitative analysis of the data shown in a. c BUN levels was decreased in Hif2a CKO mice. n = 6. Data are means ± SEM. ns means no significance. *P > 0.05, **P < 0.05, ***P < 0.001.

Fig 4: HIF2A stabilization under normoxia promotes quiescence, self-renewal, and stemness of SCs yet impedes myogenic differentiation.(A) Diagram depicting the timeline and plasmids used for HIF2A stabilization in normoxic SC culture. Pound signs denote the number and locations of point mutations in HIF2ATM. (B) Representative images of transfected (GFP+) SC clusters on myofibers from C57BL/6 mice (n >50 myofibers from 7 mice/group). The SC clusters were transfected with either HIF2ATM or control plasmids and stained for Pax7, MyoD, and DAPI. Scale bar: 5 µm. (C) Number of SC clusters, Pax7+ SCs per SC cluster, and Pax7+MyoD–, Pax7+MyoD+, and Pax7–MyoD+ SCs per SC cluster (n >50 myofibers). (D) Diagram of the experimental scheme for transplantation of HIF2A-stabilized SCs and tracing of their cell fates in vivo. (E) Cross-sectional images of TA muscles that were transplanted with HIF2ATM- or control plasmid–transfected SCs (n = 5 mice/group; 21 days after the first injury). Immunofluorescence of Pax7 and nmGFP revealed 2 fates of transplanted SCs: engrafted SCs that retained stemness (nmGFP+Pax7+; circles, bottom) and engrafted SCs that differentiated into myonuclei (nmGFP+Pax7–; arrowheads). Scale bars: 20 µm. (F and H) Percentage of engrafted and self-renewed nmGFP+Pax7+ SCs in the total SC pool after the first round (21 dpi; n = 5 mice/group in F) and second round (30 dpi; n = 6 mice/group in H) of regeneration. (G and I) Number of nmGFP+Pax7– differentiated myonuclei per TA muscle section after the first round (G) and second round (I) of regeneration. *P < 0.05, **P < 0.01, and ***P < 0.005, by 2-sided Student’s t test. Data represent the mean ± SEM.

Fig 5: Transient pharmacological inhibition of HIF2A in CTX-injured muscle promotes SC proliferation and accelerates muscle regeneration.(A) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice. (B) Representative images of HIF-c2– or 1% DMSO–treated TA muscles (3 dpi) from C57BL/6 mice (n = 6 mice/group). Immunofluorescence revealed increased Pax7+EdU+ proliferative SCs (arrowheads) and decreased Pax7+EdU– QSCs (circles). Scale bar: 20 µm. (C) Number of EdU+ and EdU– SCs per TA muscle section 3 dpi (n = 6). (D) Number of Pax7+ SCs per TA muscle section 7 dpi, 10 dpi, and 30 dpi (n = 6 or 7). (E) Representative eMyHC and laminin B2 immunofluorescence in HIF-c2– or DMSO-treated TA muscles 7 dpi and 30 dpi (n = 3). Scale bars: 20 µm. (F) Immunoblots showing the expression levels of HIF2A, eMyHC, MyoD, and tubulin in HIF-c2– or DMSO-treated TA muscles 7 dpi (n = 3). (G) Distributions of myofiber cross-sectional areas of HIF-c2– or DMSO-treated TA muscles 30 dpi (n = 3). (H) Maximal torques of uninjured TA muscles and HIF-c2– or DMSO-treated TA muscles 7 dpi (n = 6), 10 dpi (n = 3), and 30 dpi (n = 3). (I) Timeline of pharmacological inhibition of HIF2A after CTX-induced muscle injury in C57BL/6 mice and SC-HIF2AKO mice. (J)Representative Pax7 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO mice (n = 6 mice/group) 10 dpi and number of Pax7+ SCs per TA muscle section 7 dpi. Scale bar: 20 um. (K) Representative eMyHC and laminin B2 immunofluorescence images of HIF-c2– or DMSO-treated TA muscles from SC-HIF2AKO (n = 6 mice/group) 7 dpi. Scale bar: 20 µm. *P < 0.05, **P < 0.01, and ***P < 0.005, by 2-sided Student’s t test. Data represent the mean ± SEM.