Figure 4: HDAC inhibition increases HIF-2α expression in UPS.
From: Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth
(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-1α and HIF-2α 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.
