Figure 7: KLF14 overexpression induces mitotic catastrophe.

(a) HeLa cells were transfected with green fluorescent protein (GFP), GFP-KLF14 or GFP-KLF14-ΔZF2 for 36 h. Cells were stained with propidium iodide (PI) and analysed by FACS. The DNA staining profiles of GFP-positive cells were displayed. Bar graphs showing per cent cells in G2/M-phase. (b) HeLa cells were infected with indicated lentiviral constructs for 36 h. Cells were stained with mithramycin A and analysed by FACS. Bargraphs showing per cent cells in M-phase. Western blotting showing KLF14 expression probed with anti-KLF14 antibody. (c) HeLa cells were transfected with indicated plasmids for 36 h and analysed for cyclin B1 and histone 3 phosphorylation (H3-p) levels by western blotting. (d) HeLa cells were co-transfected with indicated plasmids for 36 h, then stained with PI and analysed by FACS. The DNA staining profiles of GFP-positive cells were displayed. Bar graphs showing per cent cells in G2/M-phase. (e) 293T cells transfected with indicated plasmids were harvested at indicated time points and analysed for cell viability by MTT assay. (f) HeLa cells were transfected with GFP or GFP-KLF14 for 72 h and analysed by TUNEL assay (scale bar, 50 μm). Bar graph shows quantitative data of TUNEL-positive cells. More than 100 cells per experimental group were counted. (g) HeLa cells transfected with vector (Control) or KLF14 were treated with caffeine (2 mM) or caspase inhibitor Z-VAD-fmk (20 μM) for 60 h, then stained with Annexin V–fluorescein isothiocyante (FITC) and PI, and analysed by FACS. Bar graph shows percentage of apoptotic cells. (h) HeLa cells were transfected with indicated plasmids and analysed for activated caspase-3 and Poly (ADP-ribose) polymerase cleavage by western blotting. (i) HeLa cells transfected with vector (Control) or KLF14 were treated with caffeine or Z-VAD-Fmk for 60 h, and the DNA fragmentation was analysed by agarose gel electrophoresis. MNNG (500 μM, 4 h) serves as a positive control for necrosis. (j) Plk4 co-transfection decreased KLF14 overexpression-induced cell death. HeLa cells were transfected with indicated plasmids for 60 h and analysed for cell viability by MTT assay. All experiments were repeated three times, data represent mean±s.d., *P<0.05, **P<0.01. See also Supplementary Fig. 5.