Fig. 1
ATF2-KO cells are more resistant to 5-FU therapy. a 5-FU treatment at different doses (2.5 µM, 15 µM, and 50 µM) induced p-ATF2Thr71 and ATF2 in Western blotting. ctrl: 24 h nontreated cells for control. For immunofluorescence, the cells were seeded on coverslips and then treated with 15 µM 5-FU for 48 h, and p-ATF2Thr71 was visualized by Alexa Fluor 555 goat anti-rabbit staining (red). DAPI was used for cell nuclei (blue). 20X magnification images were taken with fluorescence microscopy, scale bar: 50 μm; ctrl*: 48 h DMSO treated cells for control. b 5-FU (15 µM for 48 h) induces p-JNK and JNK in Western blotting; ctrl: 24 h nontreated cells for control, ctrl*: 48 h DMSO treated cells for control. c Representative Western blotting for intrinsic apoptotic markers Bcl-2, Bax, and caspase 9, cleaved caspase 9 in HCT116, and ATF2-KO E5 and F9 cells upon 15 µM 5-FU exposure for 48 h; ctrl: 24 h nontreated cells for control, ctrl*: 48 h DMSO treated cells for control, GAPDH was used as loading control. d Flow cytometry analysis of Annexin-PI staining in HCT116 and ATF2-KO E5 and F9 cells upon 15 µM 5-FU exposure for 48 h; percentages of alive, early apoptotic, late apoptotic and necrotic populations are given, ctrl: 24 h nontreated cells for control. e Images showing the effects of 5-FU on long-term cell viability by colony formation assay. Digitally enlarged images of 5-FU-treated plates show the presence of cells in each condition. Cells were treated for 48 h with 15 µM 5-FU or DMSO (ctrl*), t test (n = 4) *P ≤ 0.05. The normalized number of colonies was calculated against the corresponding DMSO control. Ratios for E5 and F9 were normalized against the HCT116 ratio. f Western blotting for PARP, cleaved PARP and morphologic changes upon treatment. Ratios were calculated as cleaved PARP versus noncleaved PARP (ImageJ). For visual reasons, the ratios were multiplied by 10. g, Cells were pretreated with 1 h Z-VAD (50 μM), followed by treatment with 15 μM 5-FU for 48 h; 24 h nontreated cells were used as controls (-), 48 h DMSO-treated cells were used as controls (-*), and GAPDH was used as a loading control