Fig 1: Lipid unsaturation changes with esophageal adenocarcinoma (EAC) progression, linked to desaturase proteins. (A) RNA expression of enzymes involved in lipid desaturation, taken from an independent cohort of patient biopsies representing normal esophagus (NE, n = 7), Barrett's esophagus (BE, n = 9), and esophageal adenocarcinoma (EAC, n = 8). Data are represented using boxplots, with Mann-Whitney U test or one-way ANOVA with Benjamini-Hochberg adjustment, with significance (p < .05) from NE represented by shading in the boxplots. (B) Scoring of immunohistochemistry for FADS1 and FADS2 was performed on a 4-point scale (0–3) with representative areas (indicated with black shapes) to highlight normal squamous epithelium (NSE), non-dysplastic Barrett's esophagus (BE), low-grade dysplasia (LGD), high-grade dysplasia (HGD), HGD with intraepithelial carcinoma (HGD + IEC) in 1.5 mm cores Pooled scores (n shown in brackets) are shown in (C). Effect likelihood ratio tests provided p values of < .0001 for both FADS1 and FADS2. Ordinal logistic fit provided no significant sequential changes during disease progression for FADS1, and significant changes between NSE and BE (p = .024), and LGD and HGD for FADS2 (p = .042)
Fig 2: Fatty acid desaturases (FADS) inhibition alters lipid profile and DNA damage in FLO-1 EAC cells. FLO-1 EAC cell line was treated with FADS2 inhibitor SC26196 for 48 hours before treatment with a bile acid cocktail (BAC, 1000 µM final, including an equimolar mixture of sodium salts of taurocholic acid, deoxycholic acid, glycodeoxycholic acid, glycocholic acid, and glycochenodeoxycholic acid) at pH 4 for 20 min, camptothecin (CPT, 1 µM, pH 7) for 60 min, or left untreated. (A) Dose titration of SC26196 for reduction of lipids with three or four double bonds. 500 nM SC26196 provides 80% of maximal response. (B) 500 nM SC26196 decreases lipids with three or four double bonds in lipids with two chains compared to vehicle in FLO-1 cells, and (C) in lipids with two or four double bonds in single-chain lipids. (D) Changes in lipid class were also induced by 48 h exposure to 500 nM SC26196. *, p < .05 change from control. (E) Immunofluorescence staining of ?H2AX foci (green) to visualize dsDNA damage, with the nuclei stained blue (DAPI). (F) The mean foci per cell was increased with BAC and 1 µM CPT, but SC26196 treatment decreases the mean foci per cell in BAC and vehicle. (G) Similarly, the % of cells showing more than five foci per cell was increased by BAC and CPT, while FADS inhibition by SC26196 decreased the number of positive cells in vehicle and BAC. Data from biological quadruplicates and technical duplicates. ***, p = .001
Fig 3: Effects of FADS2 knockdown on the functions of BC cell lines. (A–E) RT-qPCR and Western blotting to detect the expression of FADS2 in MDA-MB-231 and BT474 cells, ß-actin acts as control. (B–F) MTS assay was used to detect the effect of FADS2 down-regulation on cell proliferation. (C–G) Transwell cell migration assay to detect the effect of FADS2 down-regulation on cell migration ability. (D–H) Transwell cell invasion assay was used to detect the effect of FADS2 down-regulation on cell invasion ability. (I) Enrichment of EMT signaling pathway with FADS2 expression was shown using enrichment analysis of TCGA. (J) Relative expression levels of FADS2, E-cadherin, N-cadherin and Snail in the MDA-MB-231 cells transfected with Control or siFADS2, GAPDH acts as control. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 4: Association between miR-193a-5p and fatty acid desaturase 1 (FADS1) during M2 polarization of macrophages. (A) The downstream targets of miR-193a-5p as analyzed through TargetScan, miRDB, ENCORI, and miRWalk online databases. (B) The association between FADS1 and miR-193a-5p expression in hypopharyngeal carcinoma specimens was analyzed by SPSS using ENCORI online databases. R: Pearson coefficient. (C) RNA pull-down was used to present the binding between miR-193a-5p as fold enrichment. (D) Dual-luciferase analysis was performed when THP-1 cells were co-transfected with FADS1-wt and miR-539-5p mimic or FADS1-mut and miR-539-5p mimic. The activity of luciferase was detected. (E) THP-1 cells were treated with IL4 for 72 h. Western blotting was performed to detect the protein levels of FADS1 and FADS2. (F) THP-1 cells were transfected with LINC01569 siRNA or the combination of LINC01569 siRNA and miR-193a-5p inhibitor for 72 h. Western blotting was performed to detect the protein levels of FADS1 and FADS2. *P<0.05; **P<0.01.
Fig 5: Correlation analysis between FADSs and tumor-infiltrating immune cells. Correlation analysis of FADS1 (A), FADS2 (B), FADS3 (C), FADS4 (D), FADS5 (E), FADST7 (F), and FADS8 (G) with tumor-infiltrating immune cells.
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