Fig 1: Silencing Mof decreases H4K16Ac levels, and down-regulates multiple pro-fibrotic genes that are associated with H4K16Ac in IPF lung fibroblasts. Representative figures of IPF lung fibroblasts with siRNA non-targeting (NT) control or Mof. (A) Histone H4K16Ac and H4K20Me3 levels were examined by western blots with histone extracts. H4 is loading control. Mof expression in these cells is shown in B. (B) The whole cell lysate were collected for western blots (Survivin is on a separate blot, the Mof knockdown was shown in online Figure S3B); and (C) RNA were collected for real-time RT-PCR to examine pro-fibrotic genes a-SMA, Nox4, Col1A1 and survivin expression at protein (B) or mRNA (C) levels. (D) Schematic of the relative locations of the PCR primers sets used to examine the association of H4K16Ac and the promoter regions with the genes listed in E-H by ChIP assays, primer sequences are listed in Table S2. (E-H) Primary IPF lung fibroblasts with siRNA NT or Mof (treated as in A), were subject to ChIP assays. DNA was immunoprecipitated with H4K16Ac antibody. The relative levels of PCR products were represented by bars of the association of H4K16Ac with promoter region of a-SMA (E), Nox4 (F), Col1A1 (G) or Survivin (H). Quantitative PCR data were analyzed by 2??Ct method, normalized to input DNA, expressed as fold changes relative to primer set A or B. Negative control represents IgG pull-down. The values are expressed as mean ± SD from average of three independent experiments of one representative cell line. *P < 0.05, siRNA Mof vs siRNA NT control.