Stable PCNA transfection protects neutrophil-differentiated PLB985 myeloid cells from apoptosis. (A) Cytosolic PCNA expression in control (CT; pcDNA3-transfected cells) and pcDNA3PCNA-transfected PLB985 cells, as detected by Western blot analysis using the Ab5 pAbs and β-actin as loading control. (B) Quantification of PCNA expression after densitometry scanning and analysis by ImageJ software. Data are means ± SEM of six independent experiments (*, P < 0.05; Student’s t test). AU, arbitrary unit. (C–G) DMF-differentiated control or PCNA- or mutated PCNA–transfected PLB985 cells were incubated with or without 2 µg/ml gliotoxin or 10 ng/ml TRAIL for 15 h to induce apoptosis. (C) Percentage of cells in the sub-G1 phase showing DNA fragmentation after propidium iodide labeling. (D) Caspase-3 expression by Western blot analysis in DMF-differentiated PLB985 cells after gliotoxin-induced apoptosis. Data are from one representative experiment that was performed four times, yielding identical results. (E) Percentage of cells showing chromatin condensation after Hoechst labeling. (F) Percentage of cells with mitochondrial depolarization after DiOC6 labeling. (G) Percentage of cells with caspase-8 activation using a fluorescent IETD-based substrate. Data from C and E–G are means ± SEM of at least four independent experiments (*, P < 0.05; **, P < 0.01; Student’s t test).
