(5Z)-7-oxozeaenol reduces TGFβ1-induced collagen expression.

<p>A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-collagen type I and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 3 separate occasions. (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) mRNA analysis Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1 (90 pM)). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown; * = p<0.05, One-Way ANOVA).</p

Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.

<p>Genes involved with a hyperproliferative response are shown.</p><p>Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.</p

(5Z)-7-oxozeaenol reduces TGFβ1 induced gingival fibroblast proliferation.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol ((5Z)-7-oxo; 400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml-1 (90 pM)) ligand or left untreated. Cultures were grown in the presence of BrdU for up to 72 hours as described in methods. One of three representative experiments is shown; (N = 4; averages+/-SEM are shown * p<0.05 for: DMSO vs TGFβ1, (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1; ** p<0.05 for: DMSO vs TGFβ1; (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1. Two-Way ANOVA followed by Tukey's Post Hoc analysis).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 mRNA expression in human gingival fibroblasts.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. Values are expressed relative to untreated control. (N = 3; averages+/-SEM are shown; **** = p<0.0001, * = p<0.05 One-Way ANOVA).</p

(5Z)-7-oxozeaenol inhibits TGFβ1-induced mRNA expression in human gingival fibroblasts.

<p>(A) Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml^-1 (90 pM)) ligand or left untreated. Total RNA was harvested six hours later and subjected to gene expression profiling using GeneChip Human Gene 1.0 ST arrays (N = 2) as described in Methods. 147 genes were up-regulated in response to TGFβ1 (1.7 fold induction compared to DMSO control group) and 139 genes of the latter group were found to be (5<i>Z</i>)-7-Oxozeaenol sensitive. (B) Human gingival fibroblasts were treated as in (A) and subject to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown. * = p<0.05; ** = p<0.01; *** = p<0.001, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 protein expression in human gingival fibroblasts.

<p>(A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-CCN2 and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 4 separate occasions and relative CCN2 expression in response to TGFβ1 was calculated using densitometry (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) Indirect immunofluorescence analysis. Human gingival fibroblasts cultured on glass coverslips as treated as in (A). Cells were fixed and stained with an anti-CCN2 antibody and DyLight 594 conjugated secondary antibody. Cells were counterstained with DAPI to detect nuclei. Representative photographs are shown. Experiments were conducted four times, and relative fluoresce intensity ratio was calculated as described in methods (N = 4, averages+/-SEM are shown. * = p<0.05, One-Way ANOVA). (C) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-phospho-TAK1 and anti-beta actin antibodies, as indicated.</p