Figure 4:

Effects of genistein on phosphorylation of P44/42 MAPK, ERα and Shc in human uterine LM cells and phosphorylation of P44/42 MAPK in human uterine SMC.

(A) Time course of the protein expression of phosphorylation of P44/42 MAPK (P-P44/42) and total P44/42 MAPK (total P44/42) in LM cells treated with 0.3% DMSO (Control) at 0 min or 1 µg/ml of genistein in phenol red-free DMEM/F-12 containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. (B) Protein expression of phosphorylation of P44/42 MAPK (P-P44/42), phospho-ERα Serine-118 (P-ERα) and total P44/42 MAPK (Total P44/42) in LM cells treated with 0.3% DMSO (Control), 1 µg/ml of genistein (Gen), 1 µM ICI 182,780 (ICI) and 10 µM PD 98059 (PD) alone, or Gen in combination with 1 µM ICI 182,780 (ICI+Gen) or Gen in combination with 10 µM PD 98059 (PD+Gen) for 10 min. (C) Protein expression of phosphorylated Shc (P-Shc) and total Shc (Shc) in LM cells treated with 0.3% DMSO (Control) at 0 min, or 1 µg/ml of Gen at 5, 10, 15 and 30 min. (D) Time course of the protein expression of P-P44/42 and total P44/42 in SMC treated with 0.3% DMSO (Control) at 0 min or 1 µg/ml of Gen phenol red-free DMEM/F-12 medium containing 10% charcoal-stripped fetal calf serum for 5, 10, 15, 30, 60 min, 24, 72 and 168 h. The blots presented are representative examples of experiments that were performed at least three times with repetitive results. Molecular weight markers are indicated on the right of the blot. The bar graphs indicated below are the quantitative representation after densitometry of data (mean ± SD) (n = 3) of three independent experiments. Values of P-P44/42, P-ERα and P-Shc band densities in LM cells or SMC were divided by the vehicle control (set at 1), respectively. *P < 0.05 versus vehicle control containing 0.3% DMSO. a, b, c and d indicate groups which are statistically different (P < 0.05).