Fig. 4: HIF-2α expression promotes tumor-initiating and invasive propensity to chondrosarcoma cells.
From: A system-level approach identifies HIF-2α as a critical regulator of chondrosarcoma progression
a GSEA was performed for Cancer stem cell gene set using transcriptome data of SW1353 cells transfected with Ctrl or EPAS1 siRNA. NES and nominal P-value are indicated. b qRT-PCR analysis of EPAS1 transcript in SW1353 (n = 9), JJ012 (n = 4), and OUMS-27 (n = 9) cells, grown as a monolayer or spheres (left panel). Relative mRNA level of EPAS1 in primary chondrosarcoma cells (GSE47823; n = 2, right panel). c The number and size of spheres were measured in SW1353 (n = 6), JJ012 (n = 3), and OUMS-27 (n = 5) cells transduced with the indicated lentiviruses. Scale bar: 200 μm. d Quantitation of colony formation assay by SW1353 (representative images, n = 4), JJ012 (n = 6), and OUMS-27 (n = 6) cells expressing indicated shRNAs. e Quantitation of the BrdU-positive cells (green) among SW1353 (representative images, n = 3), JJ012 (n = 3), and OUMS-27 (n = 3) cells expressing indicated shRNAs. Scale bar: 50 μm. f GSEA was performed for Multicancer invasiveness signature gene set using transcriptome data of SW1353 cells transfected with Ctrl or EPAS1 siRNA. NES and nominal P-value are indicated. g Quantitation of invaded SW1353 (representative images, n = 3), JJ012 (n = 4), and OUMS-27 (n = 4) cells under indicated conditions. Scale bar: 200 μm. h Quantitation of relative gelatin degradation area by SW1353 (representative images, n = 4), JJ012 (n = 3), and OUMS-27 (n = 3) cells expressing indicated shRNAs. Scale bar: 100 μm. i Validation of small guide RNA (sgRNA):Cas9-mediated on-target cleavage of EPAS1 by T7E1 cleavage assay. The hyphen above the gel image indicates wild-type gDNA of SW1353 cells not transduced with any lentivirus. Asterisks indicate cleaved PCR amplicon fragments at the expected sizes as an indicative of successful gene editing. The percentage at the bottom of the gel image indicates expected editing efficiency. j Immunoblot of HIF-2α protein. k–o Quantitation of k sphere formation assay (n = 3), l colony formation assay (n = 5), m BrdU incorporation assay (n = 3), n transwell invasion assay (n = 6), and o gelatin degradation assay (n = 3) in SW1353 cells transduced with lentivirus harboring Cas9 and indicated sgRNAs. Data represent mean ± SEM. P-values are from two-tailed t test (b–e, g, h, k–o). i, j Full-size agarose gel and immunoblot images are provided in Supplementary Fig. 9.
