Figure 5

Inhibition of macropinocytosis, but not of clathrin-mediated endocytosis, impede mitochondrial transformation.

(A,C) Mitochondria were isolated from HeLa-DsRed2-mito cells and were incubated (300 μg/ml) with HepG2 cells (A) or healthy fibroblasts (C) pre-treated with the macropinocytosis inhibitor amiloride (30 minutes, at indicated concentration) or vehicle (DMSO) for 1 h. Following incubation, excess mitochondria were removed by three washes with PBS and membrane-bound mitochondria were removed by incubation with heparin (200 μg/ml, 30 minutes) and 3 PBS washes. Fluorescence was recorded in a plate reader and is calculated as percentage from control cells and represented as relative transformation. Error bars represent SEM, n = 3, ^*P < 0.05. (B,D) Mitochondria were isolated from HeLa-DsRed2-mito cells and were incubated (300 μg/ml) with HepG2 cells (B) or healthy fibroblasts (D) pre-treated with the macropinocytosis inhibitor EIPA (30 minutes, at indicated concentration) or vehicle (DMSO) for 1 h. Following incubation and washings (as described for A and C; see above), fluorescence was recorded in a plate reader and is calculated as percentage from control cells and represented as relative transformation. Error bars represent SEM, n = 3, ^*P < 0.05, ^**P < 0.03. (E,F) Mitochondria were isolated from HeLa-DsRed2-mito cells and were incubated (300 μg/ml) with HepG2 cells (E) or healthy fibroblasts (F), pre-treated with the clathrin-mediated endocytosis inhibitory treatments: hypertonic sucrose (0.5 M, 30 minutes), potassium depletion (as described in materials and methods section) and cytosolic acidification (25 mM ammonium chloride, 30 minutes) for 1 h. Following incubation and washings (as described for A and C; see above), fluorescence was recorded in a plate reader and is calculated as percentage from control cells and represented as relative transformation. Error bars represent SEM, n = 3, ^*P < 0.05.