Fig. 2: BAHA-LANA-PEG-CPP44 catalyst promoted H2BK120 acetylation in leukemia cells.
From: Chemical catalyst manipulating cancer epigenome and transcription
a, b Cells were treated with S32 or S34 (50 µM) for 60 min. Representative microscopic images of FITC-LANAS13K-PEG550 S32 and FITC-LANAS13K-PEG550-CPP44 S34 in the indicated cell line in two independent experiments. DNA was stained with Hoechst 33342 to visualize chromatin distribution. In (b), DAPI-negative cells were selected for observation to examine compound internalization into living cells. Scale bar, 10 µm. c, d Cytotoxicity of catalyst 1 or acetyl donor 4. THP-1 cells were treated with 1 or 4 for 6 h. The bar graph shows the mean of two independent experiments. e Immunoblot showing H2BK120 acetylation (H2BK120ac) levels of recombinant nucleosomes treated with catalyst 1, 2, or 3 (2 µM) and acetyl donor 4 (0.1 mM) for 2 h. A representative blot in two independent experiments is shown. f Immunoblot showing H2BK120ac and H2BK120 ubiquitination (H2Bub) levels in THP-1 cells treated with catalyst 1, 2, or 3 (10 µM) and acetyl donor 4 (1 mM) for 4 h. Representative blots in two independent experiments are shown. g LC-MS/MS analysis for lysine acetylation levels of histone H2A, H2B, H3, or H4 in THP-1 cells treated with catalyst 1 or 2 (10 µM) and acetyl donor 4 (1 mM) for 4 h. The acetylation yield was calculated as the ratio of acetylated lysine to (acetylated lysine + unmodified lysine) detected by LC-MS/MS, and the difference of the yield with 1 from the yield with 2 is shown. Each number on the horizontal axis corresponds to the position of lysine residue in the histone protein. The bar graph shows the mean of two independent experiments. h Immunoblot showing H2BK120ac levels in the indicated cell lines treated with catalyst 1 or 2 (10 µM) and acetyl donor 4 (1 mM) for 2 h. The loading amounts of histones were visualized with Oriole staining in (e–h). Representative blot in two independent experiments is shown.
