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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: ROS-dependent HIF1α activation under forced lipid catabolism entails glycolysis and mitophagy as mediators of higher proliferation rate in cervical cancer cells

Fig. 5

ATGL activity mediates the activation of HIF1α, responsible for mitophagy and glycolysis induction. HeLa cells were transfected with ATGL plasmid for 48 h. (a) Western blot analysis of HIF1α levels. β-Actin and ATGL were used as loading and transfection control, respectively. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL. Images are representative of six independent experiments that gave similar results. (b) Western blot analysis of HIF1α levels on nuclear extracts. Lamin B1 and LDH-A were used as nuclear and cytosolic purity control, respectively. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL for each fraction. Images are representative of three independent experiments that gave similar results. (c) Immunofluorescent analysis of HIF1α subcellular localization in HeLa cells after ATGL over-expression. Images are representative of three independent experiments that gave similar results. Nuclei were stained 10 min with 1 μg/mL Hoechst 33342. HeLa cells were transfected with ATGL plasmid for 48 h and were treated with the HIF1α inhibitor (YC-1, 100 μM) 24 h before the end of the experiment. (d) RT-qPCR analysis of BNIP3 mRNA was performed. ACTB was used as reference control. Data are shown as fold change (n = 3; * p < 0.05 as indicated). Glycolytic metabolism was monitored by: (e) Western blot analysis of HK-2 levels. β-Actin and ATGL were used as loading and transfection control, respectively; HIF1α was evaluated as YC-1 treatment control. Band intensity is indicated below the corresponding band and expressed as fold-change relative to CTRL for each fraction. Images are representative of three independent experiments that gave similar results; by (f) RT-qPCR analysis of GLUT1, MCT1 and MCT4 mRNA. ACTB was used as reference control. Data are shown as fold change (n = 3; * p < 0.05 vs ATGL). CTRL was represented by a dashed line in the bar graph. (g) Evaluation of extracellular lactate content, relative absorbance was normalized on total proteins. Data are expressed as mean ± SD (n = 3; * p < 0.05; ** p < 0.01 as indicated); (h) After over-expression of ATGL and ATGL-S47A (catalytic mutant) HIF1α 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. (I) Western blot analysis of HIF1α levels performed for HeLa cells treated 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

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