Fig 1: Effect of 17β-HSD12 on cancer cell proliferation. a Every elongation round of LCFAs consists of four steps. During the first step, the elongases of very long-chain fatty acids (ELOVL1–7) condense malonyl-CoA with a substrate fatty acyl-CoA. Subsequently, 3-ketoacyl-CoA reductase (KAR or 17β-HSD12) reduces the 3-ketoacyl-CoA to 3-hydroxyacyl-CoA, which is then subjected to dehydration by 3-hydroxyacyl-CoA dehydratases (HACD1–4). A final reduction step is performed by 2,3-trans-enoyl-CoA reductase (TER) to yield a fatty acyl-CoA elongated by two carbon atoms. b The essential PUFAs linoleic acid and α-linolenic acid give rise to a wide array of ω-6 and ω-3 FAs, respectively, through a series of elongation and/or desaturation reactions. Fatty acid desaturases (FADS1 and FADS2) and ELOVLs (ELOVL2 and ELOVL5) with distinct substrate preferences participate at different steps of this metabolic pathway. The ω-6 osbond acid (22:5) and ω-3 docosahexaenoic acid (DHA, 22:6) are produced via elongation and desaturation of docosatetraenoic acid (DTA) and docosapentaenoic acid (DPA), respectively, followed by β-oxidation in peroxisomes. c MCF7, MDA-MB-453, SUM159, and MDA-MB-231 cells were transfected with mock or 17β-HSD12 siRNA or left untreated (ctrl). (Left panel) Live measurements of cell proliferation were performed with the xCELLigence RTCA DP instrument. The cell index values (average and standard deviation of four technical replicates) from one out of three independent experiments are shown. For the SUM159 cells, the cell index was normalized to 5 h to account for small differences in seeding in this experiment. (Right panel) At 24 h, 48 h, and 72 h post-transfection with siRNAs, cell nuclei were stained with Hoechst-33342 and analyzed by high-content imaging. The values represent the mean and error bars SD from at least three independent experiments per cell line, and are normalized to the mock siRNA samples at each time point (*p < 0.05, ***p < 0.001, ns not significant)
Fig 2: Possible determinants of the differential changes after 17β-HSD12 downregulation in SUM159 and MDA-MB-231 cells. a (Left panel) SUM159 cells were cultured in Ham’s F12 nutrient mixture with insulin, or RPMI 1640 with or without insulin. 17β-HSD12 expression was downregulated with siRNAs and cell number was determined 72 h later. (Right panel) Trans-well migration assay after 17β-HSD12 silencing in SUM159 cells grown in Ham’s F12 nutrient mixture with insulin or RPMI 1640 without insulin. The statistical analyses compare mock- and 17β-HSD12-siRNA samples in each case (solid lines), as well as the difference between the mock-17β-HSD12-siRNA groups under different culture conditions (dotted lines) (mean ± SD, n = 3, *p < 0.05, **p < 0.01, ns not significant). b (left panel) mRNA expression relative to PPIA of the indicated genes in MDA-MB-231 and SUM159 cells. (Middle and right panels) mRNA expression of elongase (ELOVL5 and ELOVL7) and desaturase (FADS1 and FADS2) genes 48 h after 17β-HSD12 downregulation in MDA-MB-231 and SUM159 cells (mean ± SD, n = 4, *p < 0.05, **p < 0.01, ns not significant). c The expression of FADS1, FADS2 and ELOVL5 was analyzed by western blot 48 h after 17β-HSD12 downregulation in MDA-MB-231 and SUM159 cells. A representative blot (left panel) and analysis of band density from at least three independent experiments (right panel) is shown. Values are normalized to mock siRNA samples (mean ± SD, *p < 0.05, **p < 0.01, ns not significant)
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