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. 2019 Feb 16;22:101144. doi: 10.1016/j.redox.2019.101144

Fig. 2.

Fig. 2

TUDCA enhanced mitochondrial membrane potential through increased mitochondrial PrPClevels in CKD-hMSCs. (A) Healthy-hMSCs (n = 3) and CKD-hMSCs (n = 3) were treated with TUDCA, and pretreatment Healthy-hMSCs and CKD-hMSCs with Akt inhibitor were treated with TUDCA. We measured concentrations of PrPC by ELISA. Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. untreated Healthy-hMSCs, #p < 0.05, and ##p < 0.01 vs. TUDCA treatment of Healthy-hMSCs, $p < 0.05 vs. after pretreatment with Akt inhibitor, treatment of Nor-hMSCs, &&p < 0.01 vs. untreated CKD-hMSCs, AAp < 0.01 vs. TUDCA treatment of CKD-hMSCs. (One-way ANOVA, using Tukey's post-hoc test). (B) Western blot analysis quantified the expression of PrPC and PINK1 after treatment with TUDCA of Healthy-hMSCs and CKD-hMSCs (n = 3 sample/group). (C) The expression levels were determined by densitometry relative to VACD1. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. untreated Healthy-hMSCs, ##p < 0.01 vs. TUDCA treatment of Healthy-hMSCs, $$p < 0.01 vs. untreated CKD-hMSCs. (One-way ANOVA, using Tukey's post-hoc test). (D) Immunoprecipitates with anti-PINK1 were analyzed after treatment of TUDCA-pretreated CKD-hMSCs with si-PRNP by western blot using an antibody that recognized PrPC (n = 3 sample/group). (E) The level of PrPC, whose binding with PINK1 was normalized to that of VDAC1. Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. untreated CKD-hMSCs, #p < 0.05, and ##p < 0.01 vs. TUDCA treatment of CKD-hMSCs, $p < 0.05, and $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test). (F, G) Complex Ⅰ and complex Ⅳ activities were analyzed by an ELISA after treatment of TUDCA-pretreated CKD-hMSCs with si-PRNP. Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. untreated CKD-hMSCs, #p < 0.05, and ##p < 0.01 vs. TUDCA treatment of CKD-hMSCs, $p < 0.05, and $$p < 0.01 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test). (H and I) The positive signals of MitoSOX (H) and TMRE (I) were quantified by FACS analysis with staining after treatment of TUDCA-pretreated CKD-hMSCs with si-PRNP (n = 4 sample/group). Values represent the mean ± SEM. **p < 0.01 vs. Healthy-hMSCs, #p < 0.05 and ##p < 0.01 vs. TUDCA treatment of CKD-hMSCs, $p < 0.05 vs. TUDCA-treated CKD-hMSCs pretreated with si-PRNP. (One-way ANOVA, using Tukey's post-hoc test).