Fig. 3

FOXOs regulate Lapatinib sensitivity in mouse embryonic fibroblasts. a Western blot analysis was performed to analyse the protein levels of P-EGFR, EGFR, P-ERBB2, ERBB2, FOXO3, FOXO1 in MEFs and Foxo1,3,4^−/− MEFs. Representative western blot results are shown. Tubulin was used as a protein loading control. b The mRNA levels of EGFR, ERBB2, FOXO3, FOXO1 and FOXO4 were assessed by real-time qPCR. The RPL19 housekeeping gene transcript was used as a control to normalise gene expression. c Wild-type (WT) MEFs and Foxo1,3,4^−/− MEFs were treated with increasing concentrations of Lapatinib for 3 days and colony formation was observed for 1 week. Cells were then stained with crystal violet and absorbance measured at 592 nm (right panel). The colony formation capacity was analysed by two-way ANOVA and found to be significantly different (***p < 0.00001) from one another. d Best-fit curves were generated to determine IC₅₀ for each cell line against Lapatinib in terms of clonogenicity. Numerical data represent the average ± SEM of three different experiments (**** P < 0.0001)