Fig 1: Role of EGCG-regulated Nrf2 in hemin-induced proliferation in Caco2 cells. (A) Caco2 cells were treated with hemin (100 nM, 1 µM, and 10 µM) for 48 h. Nrf2, Keap1 and ß-actin protein expression were measured by western blot analysis (n = 4; p = 0.005: Control vs. Hemin 48 h Nrf2; p < 0.001: Control vs. Hemin 72 h Nrf2; p < 0.001: Control vs. Hemin 48 h Keap1; p < 0.001: Control vs. Hemin 72 h Keap1). (B, C) Cells were pretreated with EGCG (1 µM) for 30 min prior to hemin exposure (1 µM) for 48 h. (B) Nrf2, Keap1, and ß-actin protein expression were determined by western blot analysis (n = 4; p = 0.040: Control vs. Hemin Nrf2; p = 0.003: Control vs. Hemin + EGCG Nrf2; p = 0.012: Control vs. EGCG Nrf2; p = 0.035: Control vs. Hemin Keap1). (C) The expression of GPX2, GLRX1, and SOD2 mRNA were measured in Caco2 cells following hemin or EGCG treatment (n = 8; p = 0.014: Control vs. Hemin GPX2; p = 0.014: Control vs. Hemin + EGCG GPX2; p = 0.033: Control vs. EGCG GPX2; p = 0.027: Control vs. Hemin GLRX1; p = 0.033: Control vs. Hemin + EGCG GLRX1; p = 0.006: Control vs. EGCG GLRX1; p = 0.023: Control vs. Hemin SOD2; p = 0.027: Control vs. Hemin + EGCG SOD2; p = 0.020: Control vs. EGCG SOD2). (D, E) Cells were pretreated with trigonelline (10 µM) for 30 min prior to hemin (1 µM) or EGCG (1 µM) treatment for 48 h. (D) WST-1 levels were measured by the WST-1 cell proliferation assay (n = 7; p = 0.003: Control vs. Hemin; p < 0.001: Control vs. Hemin + EGCG; p = 0.003: Hemin + EGCG vs. Hemin + EGCG + Trigonelline). (E) The expression of CDK2, CDK4, cyclin D, cyclin E, and ß-actin protein was measured by western blot analysis (n = 4; p = 0.031: Control vs. Hemin CDK2; p = 0.049: Control vs. Hemin + EGCG CDK2; p = 0.035: Hemin + EGCG vs. Hemin + EGCG + Trigonelline CDK2; p = 0.003: Control vs. Hemin CDK4; p = 0.019: Control vs. Hemin + EGCG CDK4; p = 0.016: Hemin + EGCG vs. Hemin + EGCG + Trigonelline CDK4; p = 0.030: Control vs. Hemin cyclin D; p = 0.041: Control vs. Hemin + EGCG cyclin D; p = 0.029: Hemin + EGCG vs. Hemin + EGCG + Trigonelline cyclin D; p = 0.042: Control vs. Hemin cyclin E; p = 0.007: Control vs. Hemin + EGCG cyclin E; p = 0.004: Hemin + EGCG vs. Hemin + EGCG + Trigonelline cyclin E). Quantitative data are presented as the mean ± SE of the mean with scatter plots.WST, water-soluble tetrazolium salt; EGCG, epigallocatechin-3-gallate; Nrf2, nuclear factor erythroid-2-related factor 2.*p < 0.05.