Figure 2. RG1 enhances BM-MSCs paracrine effects on irradiated IEC-6 cells in a dose-depend manner. (A)Cell proliferation of irradiated IEC-6 cell treated with DMEM-F12, MSC-CM or CM from BM-MSCs pre-activated by different concentration of RG1 (0.1 μM, 1 μM, 10 μM, 100 μM) is detected by CCK8 from day 0 to day 7. Data represent mean ± SD of three independent experiments. **, P < 0.05 versus IR + DMEM-F12. (B) Immunofluorescence staining of IEC-6 cells with PCNA 3 days after radiation. Scale bars 100 μm. (C) Quantification of PCNA-positive cells. Data are reported as mean ± SD for 10 random fields per wells from at least three replicate wells per group. FOV, field of view. **, P < 0.05 versus IR + DMEM-F12. ##, P < 0.05 versus IR + MSC-CM (D) The protein levels of p53 in IEC-6 were detected by western blot assays 3 days after radiation with GAPDH as the internal control. Data represent the mean ± SD (n=3). **, P < 0.05 versus IR + DMEM-F12. ##, P < 0.05 versus Control. (E) Cumulative survival for rats exposed to 14 Gy abdominal irradiation by infusing with DMEM-F12, MSC-CM or RG1-MSC-CM (1 μM RG1) was analyzed using the Kaplan-Meier method. The cumulated number of rats in each experimental group is presented in parenthesis. P-values were determined by log-rank testing. (F) Mean survival time. Data represent the mean ± SD. **, P < 0.05 versus IR + DMEM-F12. ##, P < 0.05 versus IR + MSC-CM. All data were analyzed by t-test or one-way ANOVA except as otherwise indicated.