Figure 3.

PHB2 overexpression reduces apoptosis of PE_PGRS30-expressing cells. (A) Localization of overexpressed PHB2 in cells. RAW264.7 cells were transfected with PHB2-FLAG expression vector. Cells were stained with Hoechst 33342 (for nuclei), MitoTracker Red CMXRos (for mitochondria) and antibodies (for PHB2) at 24 h post-transfection. Anti-PHB2 or anti-FLAG-tag antibodies were used to detect endogenous PHB2 or overexpressed PHB2-FLAG, respectively. Scale bar, 10 μm. (B) Numbers of PE_PGRS30-expressing cells in PHB2-overexpressing cells. PE_PGRS30-Myc expression vector and PHB2-overexpression or control GFP-expression vector were transfected into RAW264.7 cells simultaneously. Numbers of PE_PGRS30-expresing cells in PHB2-overexpressing cells or in control GFP-expressing cells were counted at the indicated time points. (C) Proportions of PE_PGRS30-expressing cells with condensed nuclei in PHB2-overexpressing cells. Cells were stained with Hoechst 33342 at the indicated time points. Numbers of PE_PGRS30-expressing cells with condensed nuclei in PHB2-overexpressing cells were counted and are presented as percentages against total numbers of PE_PGRS30-expressing cells in PHB2-overexpressing cells. (D) LDH levels in culture supernatants of GFP-and PE_PGRS30-expressing cells, and PHB2-and PE_PGRS30-expressing cells. (A–D) Data are representative of three independent experiments. (B–D) Means ± SD are shown. *p < 0.05 using one-way ANOVA with Tukey’s multiple comparisons. More than 100 cells overexpressing PHB2 were counted in each experiment.