Figure 5. Aza restores EPO expression in myofibroblasts and TGF-β1–exposed pericytes.

(A) Schema showing CoCl₂ stimulation of myofibroblasts after 3-day exposure to Aza or Veh and then 2-day drug withdrawal. (B) Western blot analysis showing the inhibitory effect of 500 nM Aza on DNMT1 expression of UUO kidney myofibroblasts. A representative blot of 4 independent experiments is shown. (C) Epo 5′-UTR methylation in myofibroblasts determined by MSP using the same method as in Figure 4, D and E. Myofibroblasts were analyzed at day 5, as described in A. Representative electrophoresis of 4 independent experiments is shown. (D) Annexin V apoptosis assay of myofibroblasts at day 3 and day 5, as described in A. n = 4 per group per time point. (E) Cell cycle analysis of myofibroblasts by measuring DNA content using propidium iodide staining at day 5, as described in A. The data were means of 4 independent experiments. (F) Expression of Epo, Phd3, and Vegfa in myofibroblasts with or without CoCl₂ for 16 hours. Transient exposure to Aza was performed, as described in A. n = 4 per group. (G) Expression of Acta2 in myofibroblasts at day 5, as described in A. n = 4 per group. (H) Expression of Dnmt1, Dnmt3a, Dnmt3b, and Tgfb1 in kidney pericytes cultured in medium containing 5 ng/ml TGF-β1 or Veh for 24 hours. n = 4 per group. (I) Schema illustrating the culture of pericytes with or without TGF-β1 in the presence of 500 nM Aza or Veh. (J) Methylation of Epo 5′-UTR in pericytes determined by MSP at day 3, as described in I. Representative electrophoresis of 4 independent experiments is shown. (K) Expression of Epo, Phd3 and Vegfa in pericytes at day 3, as described in I. n = 4 per group. One-way ANOVA was used for analyses of data in D, F, H, and K, and Student’s t test was used for analyses of data in E and G. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.