Fig. 5: OA directly interacts with and stabilizes HOXB9.
From: Metabolic syndrome promotes endometrial cancer by Oleic acid-mediated polyamine accumulation
a WB of HOXB9 in Ishikawa and AN3CA cells treated with/without OA (30 µM, 48 h), adding CHX (100 μg/mL) at different times; right: HOXB9 degradation curve. Data: mean ± SEM (n = 3 independent experiments). Two-tailed two-way ANOVA. b Co-IP: Ishikawa/AN3CA cells transfected with Flag+HA-Ub, Flag-HOXB9 + HA-Ub (with/without OA 30 µM, with/without putrescine 50 µM) for 48 h, and treated with MG132 6 h before harvest; cell lysates were then incubated with anti-Flag M2 beads, blotted with anti-Praja2/ODC1. Praja2 blot grayscale: mean ± SEM ( ≥ 3 independent experiments). Two-tailed one-way ANOVA. Same-experiment samples: different gels (Praja2, ODC1, Flag; Flag, β-actin; HA), parallelled processed. c Representative Bodipy 493/503 staining of lipid droplets in AN3CA cells transfected with siNC/siODC1, with/without OA 30 µM (48 h), with/without putrescine (50 µM), spermidine(10 µM), spermine (10 µM) for 72 h (n = 3 independent experiments). Scale bars: 100 µm. d Statistical analysis of mean lipid droplet intensity in (c): Data: mean ± SD (3–6 fields; trend observed in 3 independent experiments). Two-tailed one-way ANOVA. e Triple immunofluorescence of Ishikawa/AN3CA cells treated with/without 30 μM OA (24/48 h): anti-HOXB9 (violet), anti-ODC1 (green), lipid droplets (Nile red), Hoechst (blue); visualized by HIS-SIM (n = 3 independent experiments). Scale bars: 5 µm. f Co-IP: Ishikawa cells transfected with Flag, Flag-ODC1 with/without OA (30 µM, 48 h); incubated with anti-Flag beads, blotted with anti-HOXB9. HOXB9 grayscale: mean ± SEM ((n = 3 independent experiments). Two-tailed one-way ANOVA. Same-experiment samples: different gels (Flag, HOXB9; Flag, β-actin), parallelled processed. g SPR assay of GST/GST-HOXB9/GST-ODC1 and OA; corrected response curves shown. h Thermal shift assay of HOXB9/ODC1 stability with/without 30 μM OA: GST-HOXB9/GST-ODC1 (upper) or AN3CA lysates (lower) (n = 3 independent experiments). Same-experiment samples: different gels (ODC1; HOXB9, β-actin), parallelled processed. i Working model: OA directly binds HOXB9, stabilizing it by preventing interaction with E3 ligase Praja2; HOXB9 interacts with ODC1, leading to polyamine accumulation and EC progression. Created in BioRender. Zhai, L. (2025) https://BioRender.com/e31mxj9. Multiple comparisons were corrected. Source data are provided.
