Figure 5.

Variable sensitivity of ccRCC lines to PT2399. a, c, Representative BLI of orthotopic tumors formed by UMRC-2 CMV-Luc cells (a) (vehicle, n=8 and PT2399, n=9 mice from two independent experiments) and 769-P CMV-Luc cells (c) (vehicle, n=7 and PT2399, n=8 mice from two independent experiments) before and after (30 and 35 days, respectively) treatment with PT2399 30 mg/kg or vehicle by oral gavage. b, d, Quantification of BLI as in (a) and (c), respectively. e, Immunoblots ccRCC cells lines, including quantification of HIF2α protein levels, normalized to tubulin. HIF1α in A498 and SKRC20 cells is mutated and defective¹⁴. f, NDRG1 mRNA levels in ccRCC cell lines treated with PT2399 for 24 hrs; n=3 biological replicates. For each cell line, after normalization to beta actin, NDRG1 mRNA levels were normalized to untreated value for that cell line. SLR21 VHL+/+ renal carcinoma cells were included in (e) and (f) for comparison. g, i, Immunoblots of UMRC-2 cells (g) and 769-P cells (i) after CRISPR-based gene editing with control sgRNA or HIF2α sgRNA (guides 4 and 6). h, j, Soft agar colonies formed by cells as in (g) and (i); n=3 biological replicates. Data as median with range (b, d) and mean ± s.d. (f). Statistical analysis was performed by using two-tailed Student’s t-tests (b, d). N.S., P>0.05.