Figure 3.

Afatinib promotes the pro-apoptosis ability of IR. (A and B) Flow cytometry was performed to analysis cell apoptosis of 5-8F cells (A) and HNE2 cells (B). Cells were labelled with Annexin V conjugated to FITC and propidium iodide (PI) 48 hours after IR at 4Gy or control treatment, with or without addition of 2μM afatinib as indicated. (C and D) Percent of apoptosis cells in 5-8F cells (C) and HNE2 cells (D) that were treated as described in Figure 3A-B were quantified following flow cytometry. (E) 5-8F cells and HNE2 cells were treated with IR at 4Gy and 2μM afatinib for half an hour before IR as indicated. Lysates from 5-8F and HNE2 cells were analysed via immunoblotting to detect caspase-3, cleaved caspase-3 and ACTB 72 hours later. (F) Lysates from 5-8F and HNE2 cells that were treated as described in Figure 3E were analysed via immunoblotting to detect cleaved PARP and ACTB. (G) Expression levels of caspase-3 (upper panel), Cleaved Caspase-3 (middle panel) and Cleaved PARP (bottom panel) were quantified by densitometry and normalized by ACTB levels.