Decrease of C/EBPβ protein expression attenuates DEX-induced repression of CBG mRNA and protein levels and reduces GR recruitment to the mouse <i>Cbg</i> promoter.

<p>To determine CBG mRNA and protein expression in response to decreased C/EBPβ protein expression, BWTG3 cells were transfected with non-specific siRNA (NSC) or with siRNA specific for the mouse C/EBPβ. As a control for C/EBPβ knockdown, total protein from untransfected (UT) BWTG3 cells, as well as from BWTG3 cells transfected with NSC or C/EBPβ siRNA, was harvested to perform Western blotting. A representative blot of two independent experiments is shown (A). Cells were treated with 1 nM DEX or vehicle control (0.1% EtOH) for 8 hrs, 24 hrs after transfection. (B) Relative CBG mRNA expression levels were measured by qPCR and normalized to relative GAPDH. Relative fold-expression of CBG mRNA levels were normalized to respective vehicle control (0.1% EtOH) which was set as 1. (C) CBG protein expression levels were analyzed by means of Western blotting, quantified and normalized to relative GAPDH expression. Relative CBG protein expression were normalized to the respective vehicle control (0.1% EtOH), which was set as 1. (D) A representative Western blot of CBG protein expression is shown. For (B&C) statistical analysis was done to compare CBG mRNA and protein expression in response to decreased C/EBPβ protein expression using two-way ANOVA with Bonferroni' multiple comparison as post-test (*: p<0.05; **:p<0.01; ***:p<0.001). (E) To examine the effect of decreased C/EBPβ protein expression on GR recruitment to the mouse <i>Cbg</i> promoter, BWTG3 cells were transfected with siRNA as described above. Twenty-four hrs after transfection, cells were treated with 10 µM DEX or vehicle control (0.1% EtOH) for 2 hrs followed by ChIP assay, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110702#s2" target="_blank">Material and Methods</a>. Occupancy of GR protein on the endogenous mouse <i>Cbg</i> promoter was detected using primers encompassing the regions defined as P5 to P1. Co-immunoprecipitated DNA fragments and input DNA were analyzed by qPCR and results shown are normalized to a non-specific region within the m<i>Cbg</i> promoter as well as input and expressed relative to NSC vehicle control (0.1% EtOH), which was set as 1. Statistical analysis was done to compare recruitment of the GR relative to NSC vehicle control (0.1% EtOH), using one-way ANOVA followed by Dunnett’s multiple comparison’s posttest (*: p<0.05). (F) Co-recruitment of the C/EBPβ and GR to the mouse <i>Cbg</i> promoter in response to DEX. BWTG3 cells were treated with 10 µM DEX for 2 hrs followed by re-ChIP assay, where cross-linked cell lysates were subjected to immunoprecipitation, first with an anti-GR-specific antibody (data not shown) and then with a control IgG or an anti-C/EBPβ-specific antibody (IP2). The occupancy of GR and C/EBPβ protein at the <i>Cbg</i> promoter, encompassing regions P5 to P1 were detected using primers designed against the indicated region. Co-immunoprecipitated DNA fragments and input DNA were analyzed by qPCR. Results shown were normalized to IgG for second IP as well as input and expressed relative to IgG vehicle control for the second IP (0.1% EtOH), which was set as 1. Statistical analysis was done to compare DEX-induced protein recruitment to the <i>Cbg</i> promoter relative to IgG vehicle control (0.1% EtOH), using one-way ANOVA with Dunnett’s multiple comparison as post-test (*: p<0.05).