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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

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

Fig. 3

ATGL over-expression promotes the “Warburg effect” in HeLa cells. HeLa cells were transfected for 48 h with pATGL. HeLa cells were over-expressed for 48 h and glycolytic metabolism was monitored by: (a) Western blot analysis of hexokinase-2 (HK-2), glucose transporter 1 (GLUT1) and phosphorylated pyruvate dehydrogenase (p-PDHE1α S300) levels. 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) spectrophotometric determination of hexokinase (HK) activity. Relative absorbance was normalized on total proteins and data are expressed as mean ± SD (n = 3; * p < 0.05 vs. CTRL); (c) the expression levels of glucose and lactate transporters genes through RT-qPCR analysis of GLUT1, MCT1 and MCT4. ACTB was used as reference control. Data are shown as fold change vs. CTRL represented by a dashed line in the bar graph (n = 3; * p < 0.05 vs. CTRL); (d) Evaluation of extracellular lactate content was performed on cell culture media collected after 48 h of ATGL over-expression. Relative absorbance was normalized on total proteins and data are expressed as mean ± SD (n = 3; * p < 0.05 vs. CTRL). Cells were treated with the glycolysis inhibitor 2-deoxyglucose (2DG, 10 mM) 24 h before the end of the experiment upon ATGL over-expression and (e) proliferation rate was assayed by Trypan blue direct cell counting procedure. Data are expressed as mean ± SD (n = 3; ** p < 0.01; *** p < 0.001 as indicated). (f) Cyclin D1 levels were analyzed by Western blot analysis. Band intensity is indicated below the corresponding band and expressed as fold-change relative to the untreated 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. (g) Statistical analysis of HeLa cells percentage in the different phases of the cell cycle after 48 h of ATGL over-expression determined by FACS analysis. The values represent the number of cells in each phase of the cell cycle as a percentage of the total cells. Data are expressed as mean ± SD (n = 3; * p < 0.05 as indicated)

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