Figure 1. (A) Expression of IGFBP3 gene was analyzed by qRT-PCR in MESCs treated with 200 μM H2O2 for 1 h (H2O2), or in LY-pretreated cells, stimulated with 200 μM H2O2 for 1 h, and then incubated with LY for 96 hours (H2O2+LY); Ctr – untreated cells. IGFBP3 mRNA was normalized to the reference gene SDHA, and results shown are relative to IGFBP3 expression in control cells. Data are presented as mean ± SD of duplicate determinations in two independent experiments. *p< 0.01. (B) Western blot analysis of the p-p53, PAI-1, and IGFBP3 protein expression in control (Ctr) and H2O2-treated cells. Cells were treated with 200 μM H2O2 for 1 h, and then re-cultured in fresh growth medium for the indicated time. GAPDH was used as a loading control. Representative results of three independent experiments are shown. (C) The IGFBP3 content in CM of control (Ctr) and H2O2-treated cells quantified by the ELISA. Analysis was performed at the indicated time after H2O2 treatment. Mean values ± SD of three independent experiments are shown, *p<0.01 (D) Young MESCs are heterogeneous by the level of endogenous IGFBP3 synthesis. Young cells were fixed and then stained for IGFBP3 antibodies (green), p230 (red) and Hoechst 33342 (blue). Images are presented as maximum intensity projections. Scale bars - 20 μm. Abbreviations: IGFBP3, insulin-like growth factor binding protein 3; MESCs, endometrial mesenchymal stem cells; LY (LY294002), a specific inhibitor of PI-3K; p-p53, phosphorylated p53; PAI-1, plasminogen activator inhibitor 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p230, a specific marker of trans-Golgi network; Hoechst 33342, Trihydrochloride, trihydrate, nuclear staining dye; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.