STX4 specifically regulates gene expression in foam cells.

<p><b>A</b>, Genome-wide gene expression and subsequent gene set enrichment analysis (GSEA) of macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Regulation of lipid-derived Reactome and KEGG pathways is shown. <b>B</b>, Validation of gene expression microarray analysis by quantitative PCR. For all tested samples, array observations significantly correlated with qPCR results. <b>C</b>, Gene distance matrix of genome-wide gene expression analysis of macrophages and foam cells after treatment with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Pairwise distances were calculated for comparison of two treatments and cell types. Colored squares show the distance in Euclidean space, ranging from exactly the same profile (black) to completely different (red). In macrophages STX4 expression is very different to that of T09 (P<0.0001), whereas in foam cells STX4-induced gene expression is similar to that of T09 (P<0.0001).</p

Results from competitive reporter-gene assays.

<p>Results from competitive reporter-gene assays.</p

Physiological analyses of STX4 treatment confirm unique STX4 properties in foam cells.

<p><b>A</b>, Cholesterol content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>B</b>, Cholesterol content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>C</b>, Triglyceride content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). <b>D</b>, Triglyceride content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). ***P<0.001 vs. DMSO; n.s., not significant.</p

STX4 is a novel selective LXRα agonist.

<p><b>A</b>, Chemical structure of STX4. <b>B</b>, Transcriptional activation of LXRα by T0901317 (T09), 22-R-hydroxycholesterol or STX4 in a reporter gene assay (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">Table 1</a>). Data are expressed as mean±SD (n = 3). <b>C</b>, Validation of LXRα specificity. Transcriptional activation of LXRβ by T09 or STX4 in a reporter gene assay. STX4 does not activate the LXRβ subtype (mean±SD, n = 3). <b>D</b>, Cytotoxicity of STX4 in macrophages (mean±SD, n = 3). STX4 does not reduce cellular viability up to 25 µM.</p

STX4 specifically targets diseased, oxysterol-loaden foam cells.

<p><b>A</b>, Gene expression in foam cells after siRNA-mediated, single and combined LXRα/β knockdown (mean±SEM, n = 4, fold-change vs. DMSO, logarithmised). Left, efficiency of LXRα, LXRβ and LXRα/β knockdown (kd). Middle, LXRα, LXRβ and LXRα/β knockdown (kd). Middle, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon T09 treatment. Right, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon STX4 treatment. *P<0.05, **P<0.01, ***P<0.001 vs. negative siRNA. <b>B</b>, Gene expression in THP-1 macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 4). <b>C</b>, Western blot analysis of LXRα and APOE content in foam cells and STX4 treated foam cells. Bar plot displays the results of densitometry analysis (mean±SEM, n = 3). *P<0.05, **P<0.01 vs. foam cell.<b>D</b>, Transcriptional activation of LXRα by STX4 in the presence or absence of 200 nM T09 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">table <b>1</b></a>). Data are expressed as mean±SD (n = 3). <b>E</b>, Gene expression in THP-1 macrophages after treatment with different concentrations of T09 or STX4 in the presence or absence of 1 µM T09. Data are expressed as mean±SEM (n = 2–3). *P<0.05, **P<0.01, ***P<0.001 vs. DMSO; n.s., not significant.</p

Induction of estrogenic activity of HEK293 cells transfected with ER-α receptor by compounds with selective (A) and no selective (B) toxicity against MCF-7 cells. (C) Estrogenic activity of defined compounds toward MCF-7 cell line.

<p>* <i>p</i> < 0.05 compared to the control cells treated with β-estradiol.</p

Binding site of 18 (central panel and in green in the others) and comparison with the binding mode of estradiol (Left panel, blue) and ligand AIJ (PDB accession code 1xp9) derived from the dihydrobenzoxathiin scaffold (Right panel, cyan)

<p>Binding site of 18 (central panel and in green in the others) and comparison with the binding mode of estradiol (Left panel, blue) and ligand AIJ (PDB accession code 1xp9) derived from the dihydrobenzoxathiin scaffold (Right panel, cyan)</p

Effect estrogen active compounds 15 and 18 and estrogen inactive compounds 11 and 12 on mitochondrial activity (MTT assay), cell mass (SRB assay) and LDH release in estrogen dependent MCF-7 and estrogen independent MDA-MB-231 cells.

<p>Cells were exposed to increasing concentrations of tested compounds for 72 h before performing the assay. Data are reported as % of control and are the means±SEM.</p

Estrogenic and cytotoxic activity of tested compounds in HEK293 cells transfected with wild type ERα receptor or plasmids bearing mutated versions of ERα.

<p><b>A</b>.- HEK293 were transfected with wild type ERα receptor or Erα W383A and W383S plasmids and an ER dependent luciferase reporter gene. Results are expressed as mean ± SEM (n = 8). * <i>p</i> < 0.05 was considered statistically significant. <b>B</b>.- HEK293 were transfected mock-transfected or transfected with wild type ERα or Erα W383S plasmids. 48 h after transfection, cells were incubated in the presence of increasing concentrations of compounds 18 for 72 h and cell viability was measured by MTT assay. Results are reported as % viability based on the untreated control cells normalized to 100% viable. Results represent means ± SEM (n = 8). * <i>p</i> <0.05 versus mock-transfected untransfected cells. # p<0.05 versus ERα W383S transfected cells.</p

Structures of tested compounds, all compounds possess <i>trans</i> conformation except compounds 19 and 20.

<p>Structures of tested compounds, all compounds possess <i>trans</i> conformation except compounds 19 and 20.</p