Fig. 4.
TUDCA treatment of CKD-hMSCs enhanced cell proliferation through upregulation of PrPC. (A) Cell proliferation of Healthy-hMSCs or CKD-hMSCs analyzed after treatment with or without TUDCA (n = 3 sample/group). Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. untreated Healthy-hMSCs, ##p < 0.01 vs. TUDCA-treated Healthy-hMSCs, $p < 0.05 and $$p < 0.01 vs. untreated CKD-hMSCs. (two-way ANOVA, using Tukey's post-hoc test). (B) The number of S phase Healthy-hMSCs or CKD-hMSCs was determined by FACS analysis of PI-stained cells. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. untreated Healthy-hMSCs, ##p < 0.01 vs. TUDCA-treated Healthy-hMSCs, $p < 0.05 and $$p < 0.01 vs. untreated CKD-hMSCs. (two-way ANOVA, using Tukey's post-hoc test). (C) CKD4/Cyclin D1 activity was analyzed by ELISA. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. untreated Healthy-hMSCs, ##p < 0.01 vs. TUDCA-treated Healthy-hMSCs, $p < 0.05 and $$p < 0.01 vs. untreated CKD-hMSCs. (two-way ANOVA, using Tukey's post-hoc test). (D) Western blot analysis quantified the cell cycle–associated proteins, CDK2, Cyclin E, CDK4, and cyclin D1 after treatment of CKD-hMSCs with or without TUDCA, and after treatment of TUDCA-pretreated CKD-hMSCs with si-PRNP (n = 3 sample/group). (E) The expression levels were determined by densitometry relative of β-actin. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. CKD-hMSCs, #p < 0.05 and ##p < 0.01 vs. TUDCA-treated CKD-hMSCs, $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test). (F) Cell proliferation of CKD-hMSCs analyzed after treatment of TUDCA-pretreated CKD-hMSCs with si-PRNP by a BrdU assay. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. CKD-hMSCs, #p < 0.05 and ##p < 0.01 vs. TUDCA-treated CKD-hMSCs, $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test). (G) The number of S phase CKD-hMSCs was determined by FACS analysis of PI-stained cells. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. CKD-hMSCs, #p < 0.05 and ##p < 0.01 vs. TUDCA-treated CKD-hMSCs, $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test). (H) CKD4/Cyclin D1 activity was analyzed by ELISA. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. CKD-hMSCs, #p < 0.05 and ##p < 0.01 vs. TUDCA-treated CKD-hMSCs, $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test).