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)
Fig 3: Viability and proliferation of SZ95 sebocytes after O-αSyn exposure and short-term effects on inflammation, lipid metabolism and differentiation. (a) Cell counts analysis performed in SZ95 sebocytes treated with O-αSyn (2, 5, and 10 µg/mL) for 24, 48, and 72 h. (b) Internalization of O-αSyn evaluated by immunofluorescence analysis on SZ95 cells treated with O-αSyn (10 µg/mL) for 24 h. Nuclei were counterstained with DAPI. Scale bar: 20 µm. (c) Cell cycle distribution evaluated by flow cytometric analysis on SZ95 treated with O-αSyn (10 µg/mL) for 24–48 h. The bar graph shows the distribution of cells among the different phases of the cell cycle. (d) Western blot analysis of K7, EMA, FADS2, and PPARγ in SZ95 cells treated with O-αSyn (10 µg/mL) for 48 h. Representative blots are shown. GAPDH was used as an endogenous loading control. Densitometric scanning of band intensities was performed to quantify changes in protein expression. Data are expressed as the fold change with respect to untreated control cells (control value taken as 1-fold in each case). (e) Immunofluorescence and quantitative analysis of PPARγ and BLIMP1 staining in SZ95 cells treated with O-αSyn (10 µg/mL) for 48 h. At least 450 cells for PPARγ and BLIMP1 were analyzed from three biological replicates. All the data represent the mean ± SD. Statistical significance was determined using Student’s t-test. BLIMP1: * p = 0.027 vs. untreated control. Nuclei were counterstained with DAPI. Scale bar: 50 µm.
Fig 4: Alteration of the expression of SZ95 sebocyte differentiation markers following long-term O-αSyn exposure. (a) Western blot analysis of BLIMP1, K7, EMA, FADS2, and PPARγ in SZ95 sebocytes treated with O-αSyn (10 µg/mL) for 1 week. Representative blots are shown. GAPDH was used as an endogenous loading control. Densitometric scanning of band intensities was performed to quantify changes in protein expression. Data represent the mean ± SD of three biological replicates and are expressed as the fold change with respect to untreated control cells (control value taken as 1-fold in each case) (* p < 0.05 vs. untreated control). (b) Immunofluorescence and quantitative analysis of BLIMP1, K7, EMA and PPARγ signals in SZ95 sebocytes treated with O-αSyn (10 µg/mL) for 1 week. At least 600, 700, 1000, 800 cells were evaluated for BLIMP1, K7, EMA and PPARγ, respectively. Data are presented as the mean ± SD and were obtained from three biological replicates. Statistical significance was determined using Student’s t-test. BLIMP1: ** p = 0.004; EMA: * p = 0.044; PPARγ: ** p = 0.004 vs. untreated control. Nuclei were counterstained with DAPI. Scale bar: 50 µm.
Supplier Page from Abcam for Anti-FADS2 antibody