Fig 1: AICDA is essential for KLF4 promoter demethylation. A) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1st and 7th day of differentiating BMDM from wild type and GADD45α knock out macrophages as described in Figure 3 C&D. B) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in Figure 3C&D. D) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. Figure 6A and C, * is used where ever p value is ≤0.05 and ** is used when p≤0.005. Error bars represent standard deviation.
Fig 2: Identification of the active demethylase involved in KLF4 promoter demethylation. A) PU/ER(T) cells were grown in presence of Ethanol or 100 nm Tamoxifen for the indicated time periods and cellular localization of AICDA, GADD45α, MBD4, TDG and TET2 was determined by immunoblotting with respective antibodies in cytosolic and nuclear protein fractions as described in figure 1A. B) AICDA was over expressed by electroporation of AICDA or Control plasmids in to PU/ER(T) cells and after 24 h of electroporation total AICDA and Myc in cell lysates was detected by immunoblotting. C) Under similar conditions total AICDA in cell lysates was immunoprecipitated with anti myc Rabbit polyclonal antibodies and analyzed for co-immunoprecipitation of GADD45α, MBD4, TDG and TET2 by western blotting with respective antibodies. D) Recruitment of AICDA to KLF4 promoter was assessed by ChIP in Ethanol or Tamoxifen treated PU/ER(T) cells and E) differentiating bone derived marrow macrophages on Day 1, 2 and Day 7.
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