Induction and Maintenance of Cellular Senescence

IMR90 (CCL-186) female human foetal lung fibroblasts and A549 (CCL-185) human lung adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were confirmed to be mycoplasma negative (Lonza MycoAlert, cat #LT07-118). IMR90 ER:RAS is a derivative of IMR90 cells expressing a switchable version of oncogenic H-Ras^{84 (link)}. IMR90 ER:RAS and A549 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher) supplemented with foetal bovine serum (10%), L-glutamine (2 mM, ThermoFisher), and antibiotic-antimycotic solution (1%, ThermoFisher) and incubated under standard tissue culture conditions (37 °C and 5% CO₂). For induction of the senescent phenotype, IMR90-ER:RAS cells were cultured in hydroxytamoxifen (4-OHT) (Sigma) added media at 100 nM final concentration. IMR90 were kept in culture for over 27 passages for replicative senescence. Cells were tested for mycoplasma on a regular basis.