Fig. 4

BNIP3-mediated mitophagy contributes to the increased proliferation rate as a pro-survival cell response. HeLa cells were transfected with ATGL and ATGL-S47A plasmids for 48 h. (a) Spectrophotometric determination of lipid droplets content after OIL red O staining. Relative absorbance was normalized on total proteins. Data are expressed as mean ± SD (n = 3; * p < 0.05 as indicated). (b) Proliferation was assayed by Trypan blue direct cell counting procedure. Data are expressed as mean ± SD (n = 3; * p < 0.05 vs. CTRL and ATGL-S47A). (c) HK-2 and GLUT1 levels were evaluated by Western blot analysis. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. Images are representative of three independent experiments that gave similar results. β-Actin and ATGL were used as loading and transfection control, respectively. (d) RT-qPCR analysis of Cpt1a mRNA. ACTB was used as reference control. Data are showed as fold change vs. CTRL (n = 3; * p < 0.05 vs. CTRL). (e) Reactive Oxygen Species (ROS) were quantified by cytofluorimetric analysis. Cells were treated with 50 μM of DHE 30 min before the end of the experiment. 10,000 events were counted. Data are expressed as mean ± SD (n = 3; * p < 0.05 vs. CTRL). (F) Ratio between the mean fluorescence intensity of MTR, which measures the mitochondrial potential, and of MTG, which measures the mitochondrial mass. Cytofluorimetric analysis was performed after treatment with 200 μM of MTR and MTG 30 min before the end of the experiments. 10,000 events were counted. Data are expressed as mean ± SD (n = 3; * p < 0.05 vs. CTRL). (g) Western blot analysis on mitochondrial fraction of LC3 and BNIP3 levels. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. Images are representative of three independent experiments that gave similar results. Lamin B1 and Lactate dehydrogenase A (LDH-A) were used as mitochondrial purity controls. TOM20 was used as loading control. (h) RT-qPCR analysis of BNIP3 mRNA. ACTB was used as reference control. Data are showed as fold change vs. CTRL (n = 3; * p < 0.05 vs. CTRL). (i) Western blot analysis of BNIP3 levels after treatment of HeLa cells with 2.5 mM and 5 mM caproate for 24 h. Band intensity is indicated below the corresponding band and expressed as fold-change relative to vehicle. Images are representative of three independent experiments that gave similar results. β-Actin was used as loading control. (j) After over-expression of ATGL and ATGL-S47A (catalytic mutant), BNIP3 levels were evaluated by Western blot analysis. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. Images are representative of three independent experiments that gave similar results. β-Actin and ATGL were used as loading and transfection control, respectively. HeLa cells were transfected with pATGL and BNIP3 ΔTM plasmid for 48 h and (k) Western blot analysis of cleaved caspase 3 was assayed. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. Images are representative of three independent experiments that gave similar results. β-Actin was used as loading and transfection control; ATGL and BNIP3 were used as transfection controls. The high exposure (h.e.) of BNIP3 allows to show the protein levels in CTRL and ATGL samples