Rptec tert1

Seven human RCC cell lines (786-O, CaKi-1, Caki-2, A-704, 769-P, A498, and ACHN), three human immortalized normal kidney cell lines (HEK-293, RPTEC/TERT1, and CCD1103), and a normal human fibroblast cell line (BJ) were obtained from ATCC. Two human RCC cell lines SW839 and UM-RC-2 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cell lines were maintained in the Key Laboratory of Hubei University of Arts and Science. Cells were cultured in Eagle's Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum (HyClone, UK), 1% HEPES (Life Technologies, USA), and penicillin/streptomycin in a 37°C atmosphere with 5% CO₂ and 20% O₂. Interferon-α (IFN-α, SRP4595, Sigma) was reconstituted in water. Ribociclib (HY-15777, MCE) and 5-Fluorouracil (5-FU, F6627, Sigma) were reconstituted in DMSO. For single drug treatment, ribociclib at concentrations ranging from 0.05 to 0.8 μM was added to the well. For combination studies, ribociclib, 5-FU, and IFN-α alone at one single dose, the combination of ribociclib with 5-FU, and the combination of ribociclib with IFN-α were added to the well.

RPTEC-TERT1 (ATCC) were cultured according to all manufacturer recommendations, in a 37°C incubator with 5% CO₂.

Renal proximal tubular epithelial cells (RPTEC/TERT1, ATCC CRL-4031) were maintained in DMEM/F12 (Gibco, Life Technologies, Thermo Fisher Scientific Inc, Waltham, Massachusetts, US) according to the manufacturer’s protocol with addition of 2% fetal bovine serum (FBS, Gibco). To maintain the selective pressure for immortalization 0.1 mg/ml G418 Sulfate (Calbiochem, Merck, Darmstadt, Germany) was added. Cell lines were cultured in a humidified atmosphere at 37°C and 5% CO₂. Retinal pigment epithelial cells (RPE-1/hTERT, ATCC CRL-4000) were maintained in DMEM (Gibco, Life Technologies) with addition of 8% FBS and 1 mM Sodium Pyruvate (Gibco, Life Technologies). The generation of used RPE1-hTERT tet-on Cas9 TP53^KO cells was described earlier (Benedict et al., 2020 (link)). Both cell lines were obtained recently from ATCC and experiments were performed exclusively with low-passage cell lines which were regularly tested to exclude Mycoplasma infections; follow-up authentication was performed on the basis of functional assays, gene expression patterns and cellular morphology.

The MDA-MB-231 breast cancer cell line was obtained from ATCC and cultured in DMEM with 10% FBS (Hyclone). Cells were passaged regularly when they reached approximately 70% confluency and used between p5 and p15 for all experiments. For patch clamp, 1.5×10⁵ cells were seeded in an uncoated 2 cm plastic dish 24 h prior to the experiment. For voltage sensitive dye experiments, 1.0×10⁴ cells per well were seeded in an uncoated 96-well plastic plate 24 h prior to the experiment. The renal proximal tube epithelial cell (RPTEC/TERT1) line was obtained from ATCC. RPTEC/TERT1 cells were cultured in DMEM/F-12 medium (Thermo Fisher Scientific), containing L-Glutamine and HEPES and supplemented with 25 ng/ml hydrocortisone (Sigma-Aldrich), 5 pM triiodo-L-thyronine (Sigma-Aldrich), 10 ng/ml recombinant human EGF (Thermo Fisher Scientific), 25 ng/ml prostaglandin E1 (Sigma-Aldrich), 3.5 µg/ml L-Ascorbic acid (Sigma-Aldrich), 1× ITS-G (Thermo Fisher Scientific) and 100 µg/ml geneticin (Thermo Fisher Scientific). For patch clamp and voltage sensitive dye measurements, RPTEC/TERT1 cells were seeded at 50% density into 35 mm dish and 12-well plate respectively and were grown for 3 days before starting the experiments. RPTEC/TERT1 cells up to p16 were used for all the experiments. Cells were regularly tested for mycoplasma (every 2 months) by PCR.

Human immortalized PTECs (RPTEC/TERT1, ATCC CRL-4031) are cultured per ATCC’s instructions and are used for all PT model studies up to passage 20. For gene expression analysis, human primary RPTEC (Cell Science), immortalized PTECs (RPTEC-TERT1, Evercyte) and A498 (ATCC HTB-44) renal cancer cells are used and cultured per supplier’s instructions. Human neonatal dermal fibroblasts (HNDF), GFP expressing (Angio-Proteomie) are cultured per supplier’s instructions and used up to passage 15.