Fig. 4
MiR-494 regulates lipid metabolism in HCC cells. A Representative confocal images of control (pMXs) and miR-494-overexpressing Huh-7 cells stained with Nile Red to visualize lipid droplets (LDs) accumulation. The cells were cultured for 24 h under standard conditions (RPMI), in medium without glucose, and in the presence of 2-deoxy glucose (2-DG). The Y-axis shows the quantification of LDs number per cell in each condition normalized to pMXs cells cultured in standard conditions. Mean ± SD values are reported. Two independent experiments were performed. Scale bar = 20 µm. B Representative confocal images of miR-494-overexpressing Huh-7 cells following transfection with G6pc overexpressing (G6pc over) or control (pCMV6) vectors for 24 h and stained with Nile Red. The column bar graph below shows the quantification of LDs number per cell respect normalized to control. Two independent experiments were performed. Scale bar, 20 µm. C Growth curves of control (pMXs) and miR-494-overexpressing Huh-7 cells cultured in no-glucose conditions or (D) in the presence of 2-DG. Growth curves were normalized to T0. Mean ± SD values are reported. The experiments were performed in two independent experiments in quadruplicate. E Growth curves of miR-494-overexpressing Huh-7 cells transfected with G6p-overexpressing (G6pc over) or control (pCMV6) vector and cultured in no glucose conditions or (F) in the presence of 2-DG. Growth curves were normalized to T0. Mean ± SD values are reported. The experiments were performed in two independent experiments in quadruplicate. G Real time PCR analysis of metabolic genes involved in lipid metabolism and pentose phosphate pathway in control (pMXs) and miR-494-overexpressing Huh-7 cells grown for 24 h in no glucose medium or (H) in the presence of 2-DG. Y-axis reports 2−ΔΔ.Ct values corresponding to mRNA levels. Mean ± SD values are displayed. Beta-actin was used as housekeeping gene. Real Time PCR analysis was performed in two independent experiments in triplicate. The statistical analysis was performed using two-tailed unpaired Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001