Figure 7.

Suppression of ChREBP gene by specific siRNA for 48h induces downregulation of FASN and SCD1 targets, without effect on phosphorylated ERK1/2 proteins in the AKT/Ras mouse HCC cell line. (a) Effects on the levels of the selected proteins (as assessed by Western blot analysis) are shown. β-Actin was used as a loading control. (b,c) Knockdown of ChREBP in association with the MEK inhibitor U0126 for 48h triggers downregulation of proliferation and induction of apoptosis. Cell proliferation was analyzed using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology Inc.). Apoptosis was assessed with the Cell Death Detection Elisa Plus Kit (Roche Molecular Biochemicals). (d–f) Combined administration of ChREBP siRNA and U0126 synergistically downregulates glycolysis (as assessed by LDH activity; E) and the pentose phosphate shunt (as determined by glucose 6-phosphate dehydrogenase activity; F), but only ChREBP silencing negatively affects fatty acid synthesis (D). Fatty acid synthesis was measured in AKT/Ras HCC cells by incorporation of [U-[14]C]acetate into lipids as described [13]. Specifically, AKT/Ras cells were cultured in 96-well plates at 2.0 x 10[3]/well and incubated overnight. After the addition of specific siRNAs, vehicle, or inhibitors as indicated, cells were pulse-labelled with [U-[14]C]acetate, 1 μCi/well for 48 hours. Lipids were Folch extracted and counted for [14]C. Experiments were conducted three times in triplicate. Tukey–Kramer test: at least P < 0.001 a, vs scrambled siRNA; b, vs DMSO (solvent); c, vs U0126; d, vs ChREBP siRNA. Abbreviations: FA, fatty acids; U0, U0126.