Conserved patterns of transcriptional dysregulation, heterogeneity and cell states in clear cell kidney cancer
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Raw images of Immunoblots published in "Conserved patterns of transcriptional dysregulation, heterogeneity and cell states in clear cell kidney cancer" by Lombardi O, Li R, Jabbar F, Evans H, Halim S, Lima JDCC, Browning L, Byrne H, Choudhry H, Ratcliffe PJ & Mole DR. Cell Reports 2024.
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Primary cultures were maintained in growth medium (Advanced DMEM/F12 1:1, 1X Glutamax, 1X anti-anti, 1X insulin-transferrin-sodium selenite, 4 ng/ml triiodo-L-thyronine, 100 ng/ml epidermal growth factor, 36 ng/ml hydrocortisone and 10% fetal bovine serum) and kept in physoxia (except Pt3 cultures, which were maintained in normoxia). PT2385 (Belzutifan, HY-12867, MedChem Express) was used at a final concentration of 1µM for 16 hours, or 1:1000 DMSO as a vehicle control. Cultures were analyzed at early passage (passages 1-4). RCC4 cells (gift from C.H. Buys) were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, D6429) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich F7524) and 1% 100X penicillin-streptomycin antibiotics (Gibco 15140122). Cells were reverse transfected with HIF-1 (HIF1A), HIF-2 (EPAS1), and control siPOOLs (siTOOLS Biotech) using Lipofectamine RNAiMAX Transfection Reagent (Thermofisher Scientific, 13778075) according to manufacturer’s instructions. For transfection, 2x105 cells were seeded on each 6cm dish in 3.5ml of DMEM (Sigma-Aldrich, D6429) containing 10% FBS (without antibiotic) and the final siRNA concentration for each condition was 2nM (control = 2nM controlSi; HIF-1 knockdown = 1nM HIF1Asi + 1nM controlSi; HIF-2 knockdown = 1nM HIF2Asi + 1nM controlSi; double knockdown = 1nM HIF1Asi + 1nM HIF2Asi; although the latter condition was not analyzed for the purpose of this study). After 24h, medium was replaced to fresh antibiotic-free medium. After a further 24h (48h-post transfection), cells were harvested. Cell lysates were prepared, and SDS-PAGE/Western blotting was performed as described in Lombardi et al (Cell reports 41, 111652. 10.1016/j.celrep.2022.111652). Primary antibodies used were anti-HIF-1α (BD cat no. 610959), anti-HIF-2α (in-house 190b or Cell Signaling cat no. 7096S), anti-HIF-1β (Novus Biologicals cat no. NB100‐110 or Cell Signaling cat. no. 5537S), anti-CA9 (Abcam cat no. ab15086 or Cell Signaling cat no. 5649S), anti-VHL (Cell Signaling cat no. 68547S) and anti-β-actin (Abcam cat no. ab49900).