Fig. 6. Phosphomimetic mutation of T72 enhances Bmf apoptotic activity in vitro and in vivo.

A, B Lentivirus-transduced MCF-7 (A) or MCF-10A (B) cells that inducibly express Bmf variants (including WT, T72E, S74E and S77E) were treated with −/+ doxycycline (100 ng/mL) for 24 h and cells were lysed for western blot analysis. C, D Lentiviral-transduced MCF-7 (C) or MCF-10A (D) cells inducibly expressing Bmf variants were treated with or without doxycycline for 24 h and cells were harvest for apoptosis assay using annexin V-PI staining and flow cytometry. E, F Quantitation of the numbers of CD4+ , CD8+ and B220+ splenocytes in male (E) and female (F) WT, Bmf^+/T158E and Bmf^T158E/T158E mice, respectively. Quantitative results were shown as mean ± STD from 8 mice. Significance was determined by ANOVA one-way test, *p < 0.05; **p < 0.01; ***p < 0.001. G Apoptosis analysis on splenocytes from WT and Bmf^T158E/T158E mice with or without LPS stimulation using annexin V-PI staining and flow cytometry. Quantitative results were shown as mean ± STD from 5 mice. Significance was determined by ANOVA one-way test, **p < 0.01; ***p < 0.001. H Absolute (left) or relative (right) spleen weights of WT and Bmf^T158E/T158E mice (N = 12). Significance was determined by student T test, two tailed; *p < 0.05; **p < 0.01. I Myosin V-rich P1 fraction and mitochondria fraction were separately isolated from the splenocytes of WT or Bmf^T158E/T158E mice and analyzed by western blotting.