TSA treatment increases acetylation of histone H3 associated with <i>PSG</i> gene promoters.

<p>JEG-3 cells were exposed to 150 nM TSA or 0 nM TSA (DMSO 0,015%, non-treated controls) for 18 h. A) Epifluorescence immune detection of acetyl H3 (Ac-H3) protein (green, left panels) and Höechst counterstaining (blue, middle panels). Merged images are shown on the right panels. Scale bar: 10 µm. Original magnification: 1500X. B) Western blot assay to detect whole cell content of acetyl H3 (Ac-H3) in protein extracts from JEG-3 cells treated with DMSO or TSA. β-actin protein was used for normalization of protein loading. A representative experiment of three independent assays is shown. C) ChIP analyses. TSA- and DMSO-treated JEG-3 cells were fixed with formaldehyde. DNA-protein complexes were sonicated and immunoprecipitated using anti-acetyl-H3 (Ac-H3), anti-PSG (as non related antibody, NR) or without antibodies (No Ab). Input and recovered DNA samples were amplified by conventional PCR employing primer pairs flanking positions −178/−49, −970/−551, and −1463/−977 of the regulatory region of <i>PSG</i> genes (positions are indicated relative to the <i>PSG5</i> gene). A <i>PSG5</i> coding sequence and a transcriptionally active euchromatin region of the <i>GAPDH</i> promoter were used as negative and positive immunoprecipitation controls, respectively. Results are representative of duplicated PCR reactions from two independent ChIP assays. D) Densitometric quantification of amplified products of the <i>PSG</i> gene regulatory regions normalized to the corresponding input is shown after subtraction of No Ab and NR amplification. Results are presented as mean ± SD and referred as fold change respect to DMSO control condition (arbitrarily defined as 1).