Erlotinib osi 774

For drug treatment experiments in PC9 cells, cells were plated in white-bottom 384-well plates (Falcon) at a density of 400 cells per well in 40 μL of media. For siRNA experiments, 1×10⁶ cells were plated in a 10 cm cell culture dish (ThermoFisher Scientific). 24 hours later, cells were transfected with 120 pmol of siCtrl or siUSP9X (Dharmacon) following the Lipofectamine RNAiMAX transfection procedure (Life Technologies). 48 hours later, cells were plated in 384-well plates as described above. 24 hours after cell plating, a serial dilution of erlotinib or osimertinib was performed using a D300e dispenser (Tecan). 72 hours post-treatment, 10 μL of CellTiterGlo reagent (Promega) was added to each well and luminescence was quantified on an Envision MultiLabel Plate Reader (PerkinElmer). The viable cell fraction was calculated by comparing the viability of drug-treated cells to the average viability of cells treated with DMSO only (Sigma-Aldrich), normalized by fluid volume. Curve fitting was performed using GraphPad Prism (v10.1.0). Inhibitors were obtained from SelleckChem: Erlotinib-OSI-774 (S1023) and Osimertinib-AZD92921 (S7297). Area-under-the curve (AUC) analyses were performed in Prism 10 (v10.0.3).