Extended Data Figure 9. HIF2 dependence of RCC10 cells.
a, Immunoblot analysis of anti-ARNT1 immunoprecipitates (IP) and whole cell extracts (input) prepared from RCC10 cells treated with increasing amounts of PT2399 or DMSO. Control IP without ARNT1 antibody was marked as ‘C’. b, Levels of the indicated mRNAs, normalized to beta actin, in 786-O, A498 and RCC10 cells treated with PT2399 at the indicated concentration for 24 hrs or an effective HIF2α sgRNA (sgHIF2α-6), and then normalized to cells treated with DMSO or a control sgRNA, respectively. Data as mean ± s.d.; n=3 biological replicates. c, immunoblot analysis of RCC10 cells after CRISPR based editing with HIF2α sgRNAs or control sgRNA. d, e, soft agar colonies formed by RCC10 cells as in (c); n=3 biological replicates. In (e) cells were engineered to express an exogenous sgRNA-resistant HIF2α or empty vector (EV);n=3. f, Soft agar colony counts as in (e) using ImageJ software. Colonies were counted using the following criteria: circularity range from 0.5 to 1.0 and size (pixels2) from 200 to infinity. Data shown mean ± s.e.m. Statistical significance was assessed by using two-tailed Student’s t-tests (f). *P<0.05.
g–k, p53 pathway status in ccRCC lines. g, j, Immunoblot analysis of the indicated cell lines treated for 16 hours with etoposide or vehicle. Note overproduction of p53 in RCC10 cells and off-size p53 band in UMRC-2 cells. SLR21 cells are VHL+/+. Red = PT2399 sensitive in soft agar assays. Blue = PT2399 insensitive. RCC4 cells do not form soft agar colonies and are therefore indeterminate. h, Immunoblot analysis of 786-O cells that were infected with an empty lentivirus conferring puromycin resistance and then later found to have spontaneously acquired a p53 mutation (R248W) compared to cells that retained wild-type p53. Cells were treated with PT2399 for 48 hours or with nutlin-3 (30 μM) or etoposide (20 μM) for 16 hrs. i, soft agar colony formation from cells in (h) treated with PT2399; n=3 biological replicates. k, Immunoblot analysis of parental 786-O cells that underwent CRISPR-based gene editing with a control sgRNA or HIF2α sgRNA (guide 6) (as in Extended Data Fig. 2a) then treated with PT2399 for 58 hours or treated with nutlin-3 (30 μM) or etoposide (20 μM) for 10 hrs.