Figure 4

Enhanced expression of lipogenic genes in ovarian cancer. (A) Heat map of a subset of significantly altered lipid pathway-related genes by supervised clustering (Red, overexpressed and green, downregulated genes in Sh1 and Sh2 compared to NTC cells). (B) Normalized levels of SREBP1c (SREBF1), SPHK1, PLA2G3, PLA2G4A (c-PLA2) and PPARγ mRNA by quantitative RT-PCR in NTC and Sh1 and Sh2 cells. *p < 0.05; **p < 0.01; ***p < 0.001 compared to NTC cells. (C) Western blot analysis of FASN, SREBF1, PPARγ, PLA2G3 with beta tubulin as loading control. (D) FASN activity is expressed as fold change in Sh1 and Sh2 cells compared to NTC cells. Results are means (columns) of two independent experiments made in triplicate. One-factor ANOVA was used to analyze the differences in FASN activity between each experimental condition. All statistical tests were two-sided (**p ≤ 0.01). (E) Bodipy (green) and DAPI (blue) staining of the lipid droplets in NTC, Sh1, and Sh2 cells imaged in Carl Zeiss LSM 510S confocal microscope. (F) Immunoblot analysis of HSulf-1 in Sh1 vec, and Sh1 Cl 11, where β-actin was used as loading control. (G) Bodipy (green) and DAPI (blue) staining of lipid droplets and nuclei respectively in Sh1 vec and Sh1 Cl 11 cells. (H) Bodipy staining of LDs following enhanced expression of empty vector compared to PLA2G3 construct by transient transfection in OV202NTC cells shows LDs only in PLA2G3 transfected cells.