(A) Athymic nude mice with PC9/BRc1 tumors were administered vehicle, afatinib, cetuximab, or afatinib plus cetuximab. Tumor volume was determined at the indicated times after the onset of treatment. Points, values from five mice per group; bars, SE. *, P < 0.05, for the combination of afatinib plus cetuximab versus either afatinib or cetuximab alone. (B) PC9/BRc1 cells were plated in soft agar and treated with erlotinib (E), cetuximab (C), afatinib (A), or either combination of erlotinib plus cetuximab or afatinib plus cetuximab for 8 days, after which the absorbance was measured according to the manufacturer’s protocol. Data are means ± SD of triplicates from an experiment that was repeated a total of 3 times with similar results. (C) Cells were serum starved for 12 hours prior to treatment with the indicated drugs for 8 hours, after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins.