Gene Network Analysis of Glucose Linked Signaling Pathways and Their Role in Human Hepatocellular Carcinoma Cell Growth and Survival in HuH7 and HepG2 Cell Lines

Figure 2

Serum depletion affects HepG2 cell growth and survival. (a) RTCA representative experiments (left panel) are presented by cell index normalized at time of media replacement (mean values ± SEM, ). Micrographs were taken at time of treatment 0, 2 days and 4 days later, ×10 magnification, scale bar = 50 μm. Right panels represent cell indexes obtained according to the values of cell index at time of media change (0), 12 hr, 24 hr, 48 hr, or 74 hr after media replacement. Data were retrieved from 14 RTCA independent experiments. Tendency curves show that serum removal is sufficient to alter the proliferation and/or survival of HepG2 cells within 36 hr, whatever cell density is. (b) Representative flow cytometry analysis of HepG2 cell cycle by propidium iodine incorporation (IP-A). Graphs illustrate propidium iodine incorporation for 5000 cells per cm²; significant increase in Sub-G1 (dying cells) was observed in 48–72 hr serum depleted cells. (c) Scepter cell index and size analyses of low density plated HepG2 cells (5000 cells/cm²) after serum depletion. Living cells were selected in a range of 12–35 μm and both their number and their size were reduced 48 hr after serum depletion. Inversely, the number of smaller cells representing cell death was significantly increased 48 hr after serum depletion and the mean size of living cells was significantly reduced (mean values ± SD, ;Student’s -test -value, ). (d) Gene expression was performed by real-time qPCR analysis of genes representative of hepatic phenotype and function and/or dysregulated in hepatocellulocarcinoma. mRNA quantification was normalized to hypoxanthine phosphoribosyltransferase 1 HPRT1 (mean values ± SEM, independent experiments,ANOVA test -value < 0.05). AFP: alpha feto protein; AdipoR1/R2: adiponectin receptors 1 and 2; CBX3: chromobox protein 3; CKB: casein kinase B; FABP1: fatty acid binding protein 1; HES1: hairy enhancer of Split 1; HIF1A: hypoxia responsive gene 1.

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