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. 2010 Jan 6;298(3):F617–F624. doi: 10.1152/ajprenal.00636.2009

Fig. 1.

Fig. 1.

Dot1 represses endogenous connective tissue growth factor (CTGF) mRNA expression and basal CTGF promoter activity in mouse mesangial cells (MMCs). A: overexpressed Dot1 increases H3K79 methylation states in acid extracts of histone proteins. MMCs were transiently transfected with pEGFPC3 (vector) or pEGFPC3-mDot1 (Dot1). Acid extracts of histone proteins were then prepared and assayed for H3K79 methylation states with a fluorometric assay as described in materials and methods. The H3K79me1 value for vector-transected cells was set at 1, and the other values were normalized to it; n = 3. B: overexpressed Dot1 downregulates basal CTGF mRNA expression and promoter activity in mesangial cells. MMCs were transiently transfected with pEGFPC3 (vector) or pEGFPC3-mDot1 (Dot1) as in A, and total RNA was prepared for analysis of CTGF mRNA levels by quantitative RT-PCR. *P < 0.05 vs. vector, n = 4. C: luciferase assay demonstrating that Dot1 overexpression suppresses expression of a stably incorporated CTGF promoter-luciferase construct in MMCs. A mesangial cell line harboring stably transfected pGL3Zeocin-3.8CTGF was transiently transfected with pEGFPC3 (vector) or pEGFPC3-mDot1 (Dot1). Twenty-four hours later, cell lysates were prepared, and the firefly luciferase activity of each sample was normalized to its protein content to generate the “CTGF promoter activity.” The relative luciferase activity of the vector-transfected cells was designated as 1 and utilized to determine the relative level and the significance of the other samples. *P < 0.05 vs. vector-transfected controls, n = 4. D: mRNA analysis showing siRNA knockdown of Dot1 expression in MMCs. MMCs were treated with scrambled control siRNA or Dot1-specific siRNA and then subjected to qRT-PCR analysis. *P < 0.05 vs. scrambled siRNA-transfected controls, n = 4. E: mRNA analysis showing that siRNA knockdown of Dot1 upregulates expression of endogenous CTGF mRNA expression in MMCs. MMCs were treated with scrambled control siRNA or Dot1-specific siRNA and then subjected to qRT-PCR analysis of endogenous CTGF mRNA expression. *P < 0.05 vs. scrambled siRNA, n = 3.