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.