Fig. 6
ROS are the upstream mediators of the pseudo-hypoxic response occurring after ATGL overexpression. HeLa cells were transfected with ATGL plasmid for 48 h and treated with 5 mM NAC 24 h before the end of the experiment. (a) HIF1α and BNIP3 levels were analyzed by Western blot analysis. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. The images are representative of three independent experiments that gave similar results. β-Actin and ATGL were used as loading and transfection control, respectively. (b) RT-qPCR analysis of BNIP3 levels. ACTB was used as reference control. Data are shown as fold change (n = 3; * p < 0.05 as indicated). (c) The evaluation of extracellular lactate content was performed on cell culture media collected after 48 h of ATGL over-expression by spectrophotometric analysis. Data are expressed as mean ± SD (n = 3; * p < 0.05 as indicated). (d) The proliferation was assayed by Trypan blue direct cell counting procedure. Data are expressed as mean ± SD (n = 3; * p < 0.05 vs. ATGL + NAC). (e) Cells were treated with Cycloheximide (CHX, 10 μg/mL) for 2, 4, 6 h and cobalt chloride (CoCl2, 150 μM) for 6 h before the end of experiment and HIF1α protein levels were evaluated by Western blot analysis. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL for each condition. The images are representative of three independent experiments that gave similar results. β-Actin and ATGL were used as loading and transfection control, respectively. (f) HeLa cells were treated with MG132 (2 μM) for 8 h before the end of the experiment and Western blot analysis of HIF1α and ubiquitinated proteins was determined. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. The 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 as previously described and were treated with 5 mM NAC 24 h before the end of the experiment and (g) RT-qPCR analysis of HIF1α levels was performed. ACTB was used as reference control. Data are shown as fold change (n = 3; * p < 0.05 as indicated)