Dmem and ham s f12 media

ccRCC tumor cells were cultured in a medium constituted of DMEM and Ham’s F12 media (Gibco) (v/v, 3/1 mixture), 10% fetal calf serum (Sigma), 2 mM L-glutamine (Gibco), and 100 U/ml penicillin/streptomycin (Gibco). When indicated, 10 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 0.4 μg/ml hydrocortisone (Sigma-Aldrich) or 180 μM adenine (Sigma-Aldrich) were added in the medium. Medium was renewed thrice a week.
For growth data collection, tumor cells were seeded at 1,000 cells/cm² and sub-cultured every week. All cultures were performed in flasks. Upon each passage, once per week, numbers of population doublings (PD) achieved by cultures were calculated as follows: PDs = log(N/N0)/log(2), where N0 represents the number of seeded cells and N represents the number of cells obtained after 7 days of growth.
For cell confluence data, confluence was calculated with the Incucyte S2 system (Sartorius) using the dedicated function. ccRCC cells from 3 patients were seeded at 500 cells per well in a 96-well plate and placed in the Incucyte for brightfield imaging every 4 hours for 7 days. Cell confluence was calculated automatically.

HEKs were isolated from neonatal foreskin samples obtained from healthy newborns (n = 6) after circumcision following a method described previously [4 (link),32 (link)]. Briefly, the tissue was cut into small pieces and trypsinized using 0.05% Trypsin–EDTA for 2 h at 37 °C with continuous agitation. Cells were collected every 30 min and counted using trypan blue (All from Gibco, Thermo Fisher Scientific, USA). Cells were cultured at a seeding density of 2.5 × 10⁴/cm² on a feeder layer of lethally irradiated 3T3-J2 cells. Complete keratinocyte media [DMEM and Ham’s F12 media (2:1 mixture), 10% FCS, glutamine (4 mM), penicillin–streptomycin (50 IU/mL; all from Gibco), adenine (0.18 mM), insulin (5 μg/mL), cholera toxin (0.1 nM), triiodothyronine (2 nM), and hydrocortisone (0.4 μg/mL; all from Sigma Aldrich, St Louis, MO, USA)) were added to each well. Epidermal growth factor (10 ng/mL; Austral Biologicals, San Ramon, CA, USA) was added to the culture media, starting on day 3. The media were changed every alternative day. Subconfluent primary cultures were passaged at a density of 6 × 10³ cells/cm² and cultured as above. All cultures were incubated in 5% CO₂ at 37 °C.