Fig. 5: Irreversible inhibition of EGFR induces Src activation.

a HT29 cells were pre-treated with MHYs (10 μM), gefitinib (0.25 nM), or PD168393 (2 μM) for 1 h followed by EGF (100 ng/ml) for 5 min. Total cell lysates were immunoprecipitated with EGFR antibodies and then immnoblotted with phospho-Tyr antibodies. b HT29 cells were pre-treated with gefitinib at indicated concentrations and time points to measure Src phosphorylation. c HT29 cells were pre-treated with PD168393 at indicated concentrations and time points. Cells were treated with MHYs (10 μM) for 24 h. d HT29 cells were pre-treated with MHYs (10 μM) or PD168393 (2 μM) for 1 h and then were washed twice in warm SFM to remove MHYs or PD168393. This process was repeated every 2 h until 6 h from the first wash. Cells were then stimulated with EGF (100 ng/ml) for 5 min. SFM serum-free medium. e HT29 and SW620 cells were treated with MHYs (10 μM) for 24 h to measure Src phosphorylation. Relative density measurements correspond to the intensities of the immunoblotting bands normalized to an internal control (n = 3). Data are shown as mean ± SD. **p < 0.01, ***p < 0.001. f SW620 cells were treated with MHYs (10 μM) for 24 h and their viabilities were measured by MTT assays (n = 3). Data presented as a percent of control cells. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test. The ns indicates that the comparison was not statistically significant