(A) A431 GR cells were treated with control, gefitinib (1 μM), AEW541 (1 μM), or gefitinib and AEW541 at the indicated concentrations in either full serum (10% FBS) or low serum (0.5% FBS) for 6 hours. The cells were lysed and Western blots were probed with the indicated antibodies. (B) The A431 GR cells were treated with single-agent gefitinib, AEW541, Mk-0646, or combinations of gefitinib and AEW541 or gefitinib and Mk-0646 at the indicated concentrations for 6 hours. Cells were lysed as in Figure 2A, and the extracts were immunoprecipitated with an anti-p85 antibody. IPs were probed with the indicated antibodies. Extracts from the same lysates were probed with antibodies against p-Akt (Ser473) and total Akt. (C) A schematic depicting the 2 pathways leading to PI3K/Akt signaling in A431 GR cells: the EGFR/ErbB-3 and the IGFIR/IRS-1 pathways. (D) A431 GR cells were grown in Matrigel with or without gefitinib (1 μM), AEW541 (1 μM), Mk-0646 (10 μg/ml), or combinations of these drugs as specified. Photographs of the colonies were taken after 10 days. Original magnification, ×10. Right panel shows cell numbers from Matrigel experiments. Cells were harvested by trypsinization and then counted. Cell numbers are represented as percentages of untreated cells. Bars represent the mean ± SD of 3 wells. (E) A431 GR cells were grown in 12-well plates in 0.5% FBS–containing medium for 72 hours with or without drugs (same concentrations as in D) and harvested by trypsinization. Cell numbers were determined with a Coulter Counter. Bars represent the mean ± SD of 3 wells. Student’s t test was used for statistical comparisons.