Figure 3.

Analyses of target genes regulating phospholipids synthesis. A, overview of mitochondrial respiration and the contributory role of mitochondrial phospholipids synthesis. B, expression of genes regulating phospholipids synthesis in HCT116, HCT116^HIF-1α-/-, HCT116^HIF-2α-/-, HCT116^{HIF-1α-/-HIF-2α-/-}, and HCT116^{WT KRAS} cell lines. c, clone. ACSL5, acyl-CoA synthetase 5; AGPAT7, 1-acyl-sn-glycerol-3-phosphate acyltransferase 7; and PCK2, phosphoenolpyruvate carboxykinase 2 relative to β-actin were measured by real-time reverse transcription-PCR (n = 5). Bars, stdev. p < 0.05 by Student's t test comparing knockout cells with parental HCT116 cells. C, Expression of ACSL5, AGPAT7, and PCK2 in primary colon cancers. Gene expression, relative to β-ACTIN, in normal mucosa and primary tumor was determined by real-time RT-PCR. Log expression values were graphed as box and whiskers plots; showing median expression (horizontal line), surrounded by the first and third quartiles of those values (box), and the extreme values (whiskers). Hypothesis testing was performed using the Wilcoxon signed-rank test, with * = p < 0.05 considered statistically significant, comparing tumor to mucosa. D, western blot of ACSL5 in cell lines, with antibody to α-tubulin as loading control. c, clone. E, ACSL5 promoter activity in HCT116, HCT116^HIF-1α-/-, HCT116^HIF-2α-/-, and HCT116^{HIF-1α-/-HIF-2α-/-} cells (n = 5). Diamond represents HRE site. The pGL3pro construct is a minimal promoter Firefly luciferase reporter. The ACSL5-pGL3pro construct contains an 1883 bp fragment of the 5' untranslated region of the ACSL5 gene with the HRE site. This construct is subjected to site-directed mutagenesis at the HRE site to generate the ACSL5(-HRE)-pGL3pro. Bars, stdev. p < 0.05 by Student's t test comparing transfection with pGL3pro construct versus ACSL5-pGL3pro or ACSL5(-HRE)-pGL3pro constructs.