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. Author manuscript; available in PMC: 2015 Feb 9.
Published in final edited form as: Stem Cells. 2014 Jun;32(6):1437–1450. doi: 10.1002/stem.1707

Figure 3. Increased p300 HAT activity underlies the reprograming of HMECs into TICs.

Figure 3

(a) Immunoblot analysis of p300 in normal mammary MCF10A and 184hTERT cell lines compared to HTRZ, HTRY, HTRY-LT #1, and HTRY-LT #2 cells and the BLBC cell line MDA-MB-231. (b) Immunoblot analysis of p300 and PI3K signaling components in HTRY cells treated for 24 hours with DMSO, LY294002 (LY, 20 μM), or LY in combination with MG132 (MG, 5 μM). (c) Immunofluorescence of p300 localization in HTRZ and HTRY cells. Arrows denote p300-positive nuclei (visualized by DAPI). Scale bar represents 100 μm. (d) Immunoblot analysis of cytoplasmic and nuclear fractions following siRNA-mediated p300 knockdown (5 nM) in HTRY cells. CREB and vinculin were used to assess the purity of the nuclear and cytoplasmic fractions, respectively. (e) HAT activity in nuclear lysate from HTRZ, HTRY, and HTRY cells treated for 96-hours with siRNA targeting EP300. 184hTERT and MDA-MB-231 were a negative and positive control for p300 activity, respectively. (f) Histone H3 (HH3) immunoprecipitation followed by immunoblotting to measure acetylated lysine residues. Protein levels were evaluated by densitometry (normalized to HTRZ). (g) ChIP targeting the promoters of BMI1, CD44, and CD49f in induced HTRZ and HTRY cells. DNA templates were pulled down with acetyl-histone H3 (Lys9) or nonimmune IgG antibody. GAPDH served as a control. (h) Immunoblot analysis of TIC markers in HTRY cells treated with EP300 siRNA for 96 hours. (i) qRT-PCR analysis of mRNA transcript in HTRY cells pre-treated with DMSO or anacardic acid (AA) for 4 hours prior to induction. (j) HTRY cells serially passaged as mammospheres in the presence of DMSO or AA. Primary mammospheres are shown. Scale bar represents 200 μm. Data represented as mean ± SEM. P values were determined using t test. *, P <0.05; **, P < 0.01.