Fig. 3.

The anti-tumor mechanisms of afatinib in ESCC cell lines and PDXs. a Six ESCC cell lines were treated with 0, 10 nM, 100 nM, and 1 μM afatinib and harvested after 48 h. Immunoblots show the response of EGFR downstream signaling molecules to afatinib. b IHC staining for pERK, pS6, and Ki-67 in seven PDXs tumors after 21 days treatment. Representative images and interpretation (by two independent pathologists) are shown (× 200 magnification; scale bars = 100 μM). c–h KYSE450, KYSE140, and KYSE510 cells were treated with 0, 10 nM, 100 nM, and 1 μM afatinib after serum-starvation for 12 h. After 48 h of treatment, the cells were harvested and assayed as described below. The effects of afatinib on cell cycle distribution were assessed using flow cytometry after PI/RNase staining (c). The distribution of cells in the cell cycle is depicted (d). G1 phase-associated proteins (P21, P27, CDK4, CDK6, and CCND1) were assessed using western blotting (e). Flow cytometry showed the apoptosis induced by afatinib treatment using PE-annexin V and 7-AAD staining (f). The percentage of cells in early apoptosis (Q3) and late apoptosis (Q2) was calculated as the total apoptosis ratio (g). Apoptosis-related proteins (c-PARP, c-caspase8, BCL2, and BAX) were measured by western blotting after afatinib treatment (h). Data are presented as means ± SDs of three independent assays. P values were calculated using one-way ANOVA or unpaired two-tailed t tests.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant