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).</p

Primer sequences used in conventional PCR for ChIP assays and their corresponding amplification product sizes.

<p>Primer sequences used in conventional PCR for ChIP assays and their corresponding amplification product sizes.</p

TSA stimulates Sp1 acetylation and KLF6 nuclear localization.

<p>A) Protein extracts prepared from JEG-3 cells exposed to TSA or DMSO (control condition) for 18 h were subjected to western blot analysis using anti-Sp1 (upper panel), anti-KLF6 (middle panel), and anti-β-actin (lower panel) antibodies. Representative blots are shown of at least three independent experiments with similar results. B, C) Immunofluorescence analyses of endogenous Sp1 and KLF6 (green signal, left panels) distribution in DMSO-cultured JEG-3 cells (upper panels) and TSA-treated cells (lower panels). Nuclei were counterstained with Höechst dye (blue signal, middle panels). Merged images are shown on the right panels. The images presented are representative of three independent experiments. Scale bar = 10 µm. Original magnification = 1000X. D) Western blot analysis of the endogenous Sp1 and KLF6 protein content detected in the cytoplasmic/membrane (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions of JEG-3 cells treated with DMSO (lanes 1 and 3) or TSA (lanes 2 and 4). GAPDH and Ku80 protein levels were used as loading and fractionation controls. One representative experiment of four independent assays with consistent results is shown. E) Immunoprecipitation and western blot assays were performed to determine the acetylation state of overexpressed Sp1 and KLF6 in JEG-3 cells treated or not with TSA. Whole cell lysates were first precipitated with anti-Sp1 or a mix of anti-KLF6 antibodies and then subjected to western blotting with anti-acetyl lysine antibody (Ac-Lys). The same amount (30 µg) of immunoprecipitated proteins were loaded in each lane. The blots of one representative of two different experiments are shown. The ratio of acetylated (Ac) to total Sp1 or KLF6 of each independent experiment is indicated below each band.</p

TSA enhances Sp1- and KLF6-induced <i>PSG5</i> promoter activity through the CPE element.

<p>Cells co-transfected with the UB5luc (A) or CPEmutUB5luc (B) construct and the Sp1 (lanes 2 and 5), KLF6 (lanes 3 and 6) or empty (lanes 1 and 4) expression plasmids were cultured in the presence of DMSO (lanes 1–3) or 150 nM TSA (lanes 4–6). Data represent relative luciferase activity (RLA) referred to that of each reporter construct co-transfected with the corresponding empty vector (EV) and treated with DMSO. Results are shown as mean ± SEM of triplicates of two to four independent assays. Different letters on the bars indicate statistically different values (p≤0.05, Fisher test following one-way ANOVA). CPE binding sequence (uppercase bold letters) and mutated nucleotides (lowercase letters) are depicted.</p

Total PSG protein and mRNA expression is stimulated by HDAC inhibitors.

<p>A) JEG-3 cells were cultured in the presence of the indicated concentrations of NaBu (white bars), TSA (grey bars) or vehicle alone (0, control condition) for 18 h. MTT assay was used to determine cell survival. Results represent the mean ± SEM of three independent experiments performed in sixtuplicates and are shown as percentage respect to the non-treated cultures established as 100%. No statistically significant differences (p>0.05) compared to the control condition were detected. B) Total PSG mRNA levels were determined by real time RT-PCR (ABI 7500, Applied Biosystems) in JEG-3 cells cultured in the presence of the specified concentrations of NaBu (white bars) or TSA (grey bars) and in control cultures (0). Results were normalized to PPIA and expressed according to the 2^–ΔΔCt method using as calibrator the mRNA level obtained from the corresponding control cultures. Data are presented as mean ± SEM of four independent experiments performed in triplicates. Significant differences were set at * p≤0.05, respect to control. C, D) Immunofluorescence of PSG (green, left panels) and nuclear staining with Höechst (blue, middle panels) in JEG-3 cells after treatment with the indicated amounts of NaBu (C), TSA (D) or vehicle (control). Right panels: merged images. Bar = 10 µm. Original magnification: 1000X. E, F) Western blot detection of PSG protein in JEG-3 cell total extracts (E) and secreted PSG levels in culture supernatants (F) after 150 nM TSA or DMSO exposure. β-actin (for PSG cellular content) and Ponceau staining (for secreted PSG) were used as loading normalizers. A representative experiment of two independent assays is shown.</p

Induction of <i>PSG3</i> and <i>PSG5</i> promoter activity in TSA-exposed JEG-3 cells.

<p>Scheme of reporter constructs bearing the 5′ regulatory region of <i>PSG3</i> (A) and <i>PSG5</i> (C) genes. Circles depict the CPE regulatory element. Luciferase activity was determined in JEG-3 cells incubated with 150 nM TSA (grey bars) or DMSO (white bars). Results are expressed as relative luciferase activities (RLA) referred to the promoter activity of NB3luc (B) or UB5luc (D) construct in DMSO control cultures, arbitrarily set as 1. Data are shown as mean ± SEM of four independent experiments performed in triplicates. Italic bold numbers represent fold increase in luciferase activity of each construct in TSA-exposed cells relative to the control ones. Data were subjected to analysis of variance (one-way ANOVA) followed by Fisher's test to determine significant statistical differences (p≤0.05). Different letters on the right side of each bar indicate statistically different values.</p

Effect of HAT co-activators on <i>PSG5</i> promoter activation mediated by Sp1 and KLF6.

<p>JEG-3 cells were transfected with the UB5luc plasmid and the expression vectors coding for Sp1 (A) or KLF6 (B) alone (lane 1) or together with either wild-type (lanes 2, 4, and 6) or HAT-deficient (lanes 3, 5, and 7) versions of p300 (light grey bars), CBP (dark grey bars) or PCAF (black bars). Data are shown as relative luciferase activity (RLA) to that of JEG-3 cultures co-transfected with the corresponding empty vectors defined as 1 (dashed line). Results are presented as mean ± SD of one representative experiment of two independent assays performed in triplicates, with consistent results. Statistical differences (p≤0.05) were identified using two-sided Student’s t-test. * depicts statistically different values compared to that of the culture co-transfected with the corresponding empty vectors. † represents statistically different values compared to that of the culture co-transfected with the wild type version of the corresponding co-activator. ‡ shows statistically different values compared to that of the culture co-transfected with the Sp1 or KLF6 expression vector alone.</p

KLF6 protein expression throughout trophoblast cell differentiation.

<p><b>A-</b> Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). <b>B-</b> Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. <b>C-</b> Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. <b>D-</b> Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.</p

Primer sequences and concentrations used in RT-PCR assays.

<p>Primer sequences and concentrations used in RT-PCR assays.</p

KLF6 transcript and protein levels increase during trophoblast cell differentiation.

<p><b>A-</b> KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems) in CTB cells isolated from three to eight normal term placentas, and cultured in differentiating medium during the indicated times. Results were normalized to cyclophilin A and expressed according to the 2^−ΔΔCt method using as calibrator the expression level at 0 h. Results are depicted as boxplot graphs representing the medians (horizontal bars), the 25–75th percentile interquartile range (box limits), and the lowest and highest values (whiskers) of three to eight experiments performed in triplicates. Inter-group comparisons were made using the Kruskal-Wallis one-way Analysis of variance (ANOVA) with the Dunn's multiple comparisons post-hoc test of statistical significance. *p<0.05 <i>vs</i> 0 h, #p<0.05 <i>vs</i> 2 h. <b>B-</b> Protein extracts (60 µg) prepared from CTB cells cultured for the indicated hours were subjected to western blot analysis using anti-KLF6 and α-tubulin antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022438#s4" target="_blank"><i>Materials and Methods</i></a>. A representative blot is shown. The bar graph represents the densitometric quantification of KLF6/α-tubulin ratio of three independent experiments expressed as mean ±SEM. *p<0.05 <i>vs</i> 0 h, ‡p<0.05 <i>vs</i> 16 h. (Kruskal-Wallis, Dunn's).</p