Fig. 3: HOXB9 mediates OA induced ODC1 stabilization.
From: Metabolic syndrome promotes endometrial cancer by Oleic acid-mediated polyamine accumulation
a ODC1 mRNA in Ishikawa/AN3CA (Flag/Flag-HOXB9). Data: mean ± SEM (n = 3 independent experiments). Two-tailed t-test. b ODC1 protein in Ishikawa/AN3CA (Flag/Flag-HOXB9; n = 3 independent experiments). Same-experiment samples: different gels (ODC1/Flag; β-actin), parallel. c Upper: ODC1 in Ishikawa/AN3CA (Flag/Flag-HOXB9) + CHX (100 μg/mL, different times). Lower: ODC1 degradation curve. Data: mean ± SEM (n = 3 independent experiments). Two-tailed two-way ANOVA. Same-experiment samples: different gels (ODC1; HOXB9/β-actin), parallel. d Upper: ODC1 in AN3CA/HEC-50B (siNC/siHOXB9) + CHX (100 μg/mL, different times). Lower: ODC1 degradation curve. Data: mean ± SEM (n = 3 independent experiments). Two-tailed two-way ANOVA. Same-experiment samples: different gels (HOXB9/β-actin; ODC1/GAPDH), parallel. e AN3CA (siNC/si-1HOXB9/si-2HOXB9 ± MG132; n = 3 independent experiments). Same-experiment samples: different gels (ODC1; HOXB9/β-actin), parallel. f Co-IP: Ishikawa (24 h OA) /AN3CA (48 h OA, 30 µM) + anti-HOXB9, WB (anti-ODC1) (n = 3 independent experiments). Same-experiment samples: different gels (ODC1/HOXB9; HOXB9/β-actin; ODC1/GAPDH), parallel. g Left: Co-IP: Ishikawa/AN3CA (Flag/Flag-ODC1) + anti-Flag/ anti-ODC1, WB (anti-HOXB9) (n = 3 independent experiments). Right: Co-IP: Cells (Flag/Flag-HOXB9) + anti-Flag, WB (anti-ODC1) (n = 3 independent experiments). h Top: Schematic of GST-HOXB9/ODC1 domains. Lower: Bacterial purified GST/GST-HOXB9/GST-ODC1 + 293 T (Flag-ODC1/Flag-HOXB9), WB (anti-Flag) (n = 3 independent experiments). Gels: Coomassie-Blue staining. i Triple immunofluorescence of Ishikawa/AN3CA (anti-HOXB9 [red], anti-ODC1 [green], Hoechst [blue]; n = 3 independent experiments). White boxes: co-localization. Scale bars: 10 µm; 1 µm (enlarged). Right: Co-localization quantitation. j Co-IP: Ishikawa (GFP-HOXB9/Flag-ODC1) + anti-Flag, WB (anti-OAZ1); or (GFP-HOXB9/Flag-OAZ1) + anti-Flag, WB (anti-ODC1). Lower: OAZ1/ODC1 quantification. Data: mean ± SEM (n = 3 independent experiments). Paired two-tailed t-test. Same-experiment samples: different gels (OAZ1/Flag; GFP), parallel. k HOXB9/ODC1/SREBP1 in Ishikawa cytoplasmic/nuclear lysates (OA, 30 µM, indicated times) + ODC1/HOXB9 quantification. Data: mean ± SEM (n = 3 independent experiments). Two-tailed one-way ANOVA. Same-experiment samples: different gels (n-SREBP1/ODC1; HOXB9/Lamin B; fl-SREBP1/ODC1; HOXB9/β-actin), parallel. l Upper: IHC of ODC1/HOXB9 in EC tissues (N = 17 with LVSI/LNM, 3 shown). Scale bars:50 µm. Lower: Two-tailed Spearman correlations. m Multiplex immunofluorescence of HOXB9/ODC1/DAPI in EC Tissue microarray (TMA) (118 cancer; 17 peritumoral excluded). Scale bars: 2000 µm. Two-tailed Pearson correlation. n BzCl-polyamines in AN3CA ( ± OA 30 µM, siNC/siHOXB9/Flag/Flag-HOXB9). Data: mean ± SD (n = 3 technical replicates; trend in 3 independent experiments). Two-tailed one-way ANOVA. o Model: HOXB9 competes with OAZ1 to bind ODC1, inhibiting ODC1 degradation, causing polyamine accumulation. Multiple comparisons corrected. Source data provided.
