Hygromycin

All experiments were performed using MCF-10A cells at 30–80% confluency. MCF-10A cells (ATCC, CRL-10317) were cultured in phenol red-free DMEM/F12 (Invitrogen) supplemented with 5% horse serum (ATCC), 20 ng/mL EGF (PeproTech), 10 µg/mL insulin (Sigma-Aldrich), 0.5 µg/mL hydrocortisone (Sigma-Aldrich), 100 ng/mL cholera toxin (Sigma-Aldrich), 50 U/mL penicillin and 50 µg/mL streptomycin (Thermo Fisher). MCF-10A p53–/– cells were obtained from Horizon Discovery (HD 101-005). For starvation, cells were cultured for 2 days in starvation media (phenol red-free DMEM/F12 (Invitrogen)) supplemented with 0.5 µg/mL hydrocortisone (Sigma-Aldrich), 100 ng/mL cholera toxin (Sigma-Aldrich), 0.3% bovine serum albumin (Sigma-Aldrich), 50 U/mL penicillin and 50 µg/mL streptomycin (Thermo Fisher). To release into the cell cycle with EGF, cells were cultured in starvation media and 20 ng/ml EGF (PeproTech). RPE1-hTERT human retinal pigment epithelial cells (ATCC, CRL-4000) were cultured in DMEM/F12 plus 10% FBS and 0.01 mg/mL hygromycin B. RPE1 were starved in DMEM/F12 and released with DMEM/F12 plus 0.5% FBS.

pGRN145 vector containing hTERT sequence (ATCC) and hygromycin resistant gene was used to transfect ETCC001 primary cells. Transfection was performed using Lipofectamine 2000 (Life Technologies). 30 μg/ml of hygromycin (Life Technologies) was used to select for positively transfected cells. 1 × 10³ hygromycin-resistant cells were seeded in a 15-cm petri dish and grew for at least 2 weeks. Single cell colonies were then amplified. Telomerase activity in these cell colonies was measured using the TRAPeze Telomerase Detection Kit (Millipore). Heat inactivated samples were used as negative controls for the telomerase activity detection.