Figure 4: HDAC inhibition up-regulates Klotho via reversing the promoter hypoacetylation and increasing Klotho transcription.
(A) TSA up-regulates Klotho in kidney. Kidney lysates from control and TSA-treated mice (6 weeks) were analyzed for Klotho protein levels by Western blotting (3 randomly selected samples from each group were shown). (B) Quantification of Fig. 3A. (C) HDAC inhibition dose and time-dependent up-regulation of Klotho in renal cells. HK2 cells were treated TSA of various doses (10, 30, or 100 ng/ml) or different times (100 ng/ml for 8, 16, and 24 h), or treated with SAHA of various amounts (1, 2, 5 and 10 μM) for 24 h, and then cell lysates were analyzed for Klotho protein expression by Western blotting. (D) Luciferase assay. HK2 cells were transfected with a mouse Klotho promoter reporter (mKLp-Luc) plus a renilla luciferase plasmid control for 20 h, and then TSA of two doses (10 and 100 ng/ml) was added to the cells for additional 24 h. The cell lysates were analyzed for luciferase activities, presented as the fold changes of the reporter luciferase activities divided by that of renilla control. (E) Klotho mRNA from mouse kidney. The average levels of Klotho mRNA from control (Con), TSA, adenine (Ad) and TSA-treated adenine mice (6 weeks, n = 6) were determined by qRT-PCR. (F) ChIP assay. Mouse kidney lysates from control, TSA, adenine and TSA-treated adenine mice were cross-linked and immune-precipitated with an anti-acetylated Histone3 antibody. The immune-precipitated DNAs were further PCR-amplified with primer sets specific for mouse Klotho promoter (KLpro). The genomic DNAs served as input control. The PCR products were analyzed on a 1.5% agarose gel and visualized under UC light. Representative results were shown. (G) Semi-quantification of Fig. 4F from all mice (n = 6 in each group). Data are presented as mean ± SD. Cell-bases assays were repeated three times and the representative results were shown. *P < 0.05 versus control; #P < 0.05 versus adenine.
