Fig. 3. Nampt regulates the transcription of Nfatc1 by epigenetic remodeling of the promoter in RAW 264.7 cells.

a Nfatc1 expression is inhibited by the Nampt siRNA treatment. Nfatc1 protein expression in Nampt knocked down RAW 264.7 cells was measured by western blot. Equal amounts (20 µg) of whole cell lysates were immunoblotted for Nfatc1, Nampt, and Gapdh. Relative quantification of Nfatc1 gene expression in scrambled control and Nampt siRNA transfected RAW 264.7 cells (N = 4). b RAW 264.7 cells were co-nucleofected with scrambled control or Nampt siRNA and an Nfatc1-Luc reporter construct. Relative Nfatc1 luciferase activities were normalized to non-stimulated scrambled siRNA transfected control cells. Each determination represents the average of three independent experiments. Relative quantification of Nfatc1 mRNA following nascent RNA capture in RAW 264.7 cells nucleofected with either SC siRNA or Nampt siRNA. Transcriptional rates presented as fold loss of EU labeled control RNA. c ChIP-PCR analysis of RAW 264.7 cells nucleofected with either scrambled siRNA or Nampt siRNA. The P1 region of the Nfatc1 promoter was amplified following immunoprecipitation with the acetyl histone lysine 9 antibody and P1 and P2 promoter regions were amplified following immunoprecipitation with H3K4Me3 and H3K27Me3 antibodies. d Histone acetyltransferase activity (HAT) in scrambled control and Nampt siRNA transfected RAW 264.7 cells. *P < 0.05; **P < 0.005