Fig. 2

Associations of LEFTY with pSmad2 and ovarian hormone receptors in Em Cas. a Upper: staining by hematoxylin and eosin (HE) and IHC for LEFTY, pSmad2, ERα, and PR in Em Cas. Original magnification, ×200. Lower: IHC score for pSmad2, ERα, and PR in Em Cas. The data shown are means ± SDs. b Upper three panels: expression of LEFTY and pSmad2 proteins in Em Ca tissues by western blot assay. Lower: correlation between LEFTY and pSmad2 expression in Em Cas. Intensity of bands for endogenous LEFTY and pSmad2 proteins in upper three panels were calculated by normalization to β-actin using NIH Image software. ρ, Spearman’s correlation coefficient; p, p-value; N, number of cases. c Upper: western blot (left) and RT-PCR (right) analyses for the indicated molecules in Ishikawa cells following treatment with 0, 2, and 4 ng/mL TGF-β1 for 24 h. Middle: western blot analysis for the indicated proteins in Ishikawa cells following treatment with 2 ng/ml TGF-β1 for the times shown. Lower: Ishikawa cells were transfected with LEFTY1 and LEFTY2 reporter constructs, in addition to either cotransfection with Smad2 or TGF-β1 treatment. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. d Upper: expression of LEFTY in Ishikawa cells with either 10⁻⁸ M 17β-estradiol (E2), 10⁻⁸ M medroxyprogesterone 17-acetate (MPA), or ethanol (Et) for 24 h. Middle and lower: Ishikawa cells were transfected with LEFTY1 and LEFTY2 reporter constructs, together with either ERα/E2 (middle) or PRB/MPA (lower). The experiment was performed in duplicate