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. Author manuscript; available in PMC: 2020 Jun 15.
Published in final edited form as: Cancer Res. 2019 Oct 3;79(24):6215–6226. doi: 10.1158/0008-5472.CAN-19-1982

Figure 3.

Figure 3.

Regulation of tumor cell death by MFF-VDAC1 complex. A, PC3 cells were incubated with vehicle, cell-permeable scrambled peptide (scrambled) or cell-permeable MFF peptide #8–11 (10 μM), and MitoTracker (MT) and TMRE fluorescence reactivity was imaged continuously by time-lapse videomicroscopy. Representative images at t=0 min and t=120 min. B, The conditions are as in (A) and changes in TMRE and MitoTracker labeling were quantified at the indicated time intervals. The decrease in MitoTracker signal after treatment with MFF peptide #8–11 may reflect activation of mitophagy. C and D, PC3 (C) or BPH-1 (D) cells were treated with the indicated MFF peptides or CaCl2 and supernatants (Sup) or mitochondrial extracts (Mito) were analyzed by Western blotting. E, The indicated tumor (LNCaP, C4–2B, PC3, DU145, PC3) or normal (BPH-1, HFF) cell types were treated with increasing concentrations (0–40 μM) of cell-permeable scrambled peptide (Scr) or MFF peptide #8–11 (MFF) and analyzed for cell death after 2 h by CellTox reactivity. Mean±SD (n=3). F, Isogenic drug-sensitive (2058_S) or drug-resistant (2058_R) melanoma cells were incubated with increasing concentrations of cell-permeable MFF peptide #8–11 or cell-permeable scrambled peptide (50 μM) and analyzed for cell death at the indicated time intervals by CellTox reactivity. Mean±SD (n=2). G, PC3 cells were treated (0–50 μM) with cell-permeable scrambled peptide, WT MFF peptide #8–11 or MFF peptide #8–11 containing the double mutation Arg225Asp/Arg236Asp (DD) and analyzed for cell death at the indicated time intervals. Mean±SD (n=3). H, PC3 cells were incubated with cell-permeable scrambled peptide or cell-permeable MFF peptide #8–11 (0–50 μM), mixed with the indicated concentrations of cyclosporine A (CsA, 0–10 μM for 75 min), and analyzed for cell death at the indicated time intervals. Mean±SD (n=2). I, Immortalized wild type (WT) mouse embryonic fibroblasts (MEF) or CypD−/− MEF were incubated with cell-permeable scrambled peptide (Scr) or MFF peptide #8–11 (25 μM) and analyzed for cell death. Mean±SD (n=2). J, Immortalized WT or VDAC1−/− MEF were incubated with the indicated increasing concentrations of cell permeable MFF peptide #8–11 (10–50 μM) or scrambled peptide (50 μM) and analyzed for cell death at the indicated time intervals. Mean±SD (n=2).