Fig. 4.

Real-time RT-PCR studies on Msr gene expression in culture media with different pH conditions (pH 6.4, 6.8, and 7.5) at 1, 2, and 3 days in culture. All Msr genes, except for MsrB2, show increased expression at the mRNA level after 3 days of culture in acidic media with MsrB3 showing the most dramatic responses. A: MsrA mRNA levels; (B) MsrB1 mRNA levels; (C) MsrB2 mRNA levels; (D) MsrB3 mRNA levels. E: Western blotting assays on MsrA protein and β-actin after culturing MESCsin media with different pH for days 1, 2, and 3. F: Densitometry of MsrA bands normalized by β-actin after Western blotting shows equal levels of MsrA protein expression in cells cultured in media with different pH on the same days. In all comparisons, data from cells cultured at pH 7.5 are arbitrarily set as unit 1. Results with statistically significant differences from 0+0 group were labeled with asterisks (*)(P ≤0.05). Triplets were used in each experiment and at least two independent experiments were performed. Results were averaged with standard errors of mean (SEM) presented as error bars. Results are labeled with asterisks (*) if there are statistically significant differences (P ≤0.05) comparing samples treated with acidic culture media and cells cultured at pH 7.5.