Fig. 5: IDH mutation causes stabilization of HIF-2α in chondrosarcoma cells.
From: A system-level approach identifies HIF-2α as a critical regulator of chondrosarcoma progression
a Sanger sequencing confirmed the heterozygous mutation of IDH2 R172S (AGG > AGT) in SW1353 cells. Red arrowhead indicates the mutated site in the IDH2 sequence. b Intracellular D2HG levels in SW1353 cells harboring the indicated shRNA (n = 3). ND; not detected. c Immunoblot analysis of HIF-2α protein in SW1353 cells harboring Ctrl or IDH1 shRNA (n = 3). d Immunoblot analysis of HIF-2α protein in SW1353 cells harboring Ctrl or IDH2 shRNA (n = 4). e Sanger sequencing confirmed the heterozygous mutation of IDH1 R132G (CGT > GGT) in JJ012 cells. Red arrowhead indicates the mutated site in the IDH1 sequence. f Intracellular D2HG levels in JJ012 cells harboring the indicated shRNA (n = 3). g Immunoblot analysis of HIF-2α protein in JJ012 cells harboring Ctrl or IDH1 shRNA (n = 7). h Immunoblot analysis of HIF-2α protein in JJ012 cells harboring Ctrl or IDH2 shRNA (n = 3). i Cycloheximide (CHX) chase analysis of HIF-2α protein in JJ012 cells harboring Ctrl or IDH1 shRNA. Immunoblots against HIF-2α and actin (left panel) and densitometric analysis of HIF-2α band intensities, normalized against HIF-2α level at the initial time point of each condition (n = 6, right panel). j Relative D2HG level in JJ012 cells treated with different concentrations of AG-881. The levels were normalized to vehicle treatment (n = 3). k Immunoblot analysis of HIF-2α protein in JJ012 cells treated with 2 μM AG-881 for 72 h. Representative images for immunoblot analysis (left panel) and relative quantification of HIF-2α protein level (n = 6, right panel). Actin was used to verify equal loading of the samples. Data represent mean ± SEM. P-values are from one-way ANOVA (c, d, f), two-tailed t test (g, h, k), or two-way ANOVA (i). NS, not significant. (c, d, g–i, k) Full-size immunoblot images are provided in Supplementary Fig. 9.
