Volume 16, Issue 4, Pages 267-272 (October 2009) The anti-proliferation mechanism of glucocorticoid mediated by glucocorticoid receptor- regulating gene expression  Jian Lu  Pathophysiology  Volume 16, Issue 4, Pages 267-272 (October 2009) DOI: 10.1016/j.pathophys.2009.02.009 Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Dex-induced expression of TβRII mediated by GR in PC-3 cells. (A) PC-3 cells were treated with 1–100nM Dex and/or 1μM RU486, an antagonist of GR for 16h. TβRII mRNA was determined by RT-PCR. (B) TβRII protein levels of PC3 cells treated with 100nM Dex or the vehicle for different periods of time were determined by Western blot. These data are representative of the results from three independent experiments. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Dex enhances TGF-β1-induced reporter gene expression. PC-3 cells were transiently transfected with a TGF-β1/Smad-responsive promoter construct (p3TP-Luc). Twelve hours later, cells were treated with 100nM Dex or 10ng/ml TGF-β1 for another 24h and then harvested for detecting luciferase activity. The columns indicated means±S.D. for 3TP-Luc activity relative to the level in vehicle-treated control cells (Con), which was set at 1.0. p<0.05. *Statistically significant difference between a treated group and the control. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 TβRII-neutralizing antibody blocks inhibitory effect of Dex on the growth of PC-3 cells. PC-3 cells were pretreated with 20μg/ml TβRII neutralizing antibody or control IgG for 3h, and then treated with 10ng/ml TGF-β1 or 100nM Dex. Cells were allowed to grow for 6 days with refreshing the medium every other day and determined cell proliferation by MTT reduction method. Results are expressed as mean±S.D. of quadruple determinations. *Significant inhibitory effect (p<0.05) of TGF-β or Dex on the growth of PC-3 cells. #Significant blocking of the inhibitory effect (p<0.05) of TGF-β or Dex on the growth of PC-3 cells by TβRII neutralizing antibody. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Dex up-regulates RhoB expression in HO-8910 cells and other human cancer cell lines. HO-8910 cells were maintained in serum free RP1640 for 24h and then treated with 100nM Dex for different time intervals for detection of RhoB mRNA expression by semi-quantitative RT-PCR (A) and protein expression by Western blotting (B). 3AO cells, SMMC-7721 cells and HOS-8603 cells were treated with 100nM Dex for 24h followed by analysis of RhoB protein expression (C). The blots reflect one study that is representative of three independent experiments. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 RhoB alters the growth effect of Dex on HO-8910 cell lines. Cells were stably transfected with each RhoB plasmids or control vectors (pcDNA3, RNAi-control) and then were selected with 400μg/ml of G418 for 1 month. Then cells were cultured in medium containing 5% CD-CBS in the presence or absence of 10 or 100nM Dex for 6 days, and assessed the cell proliferation ability by MTT assay. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 RhoB enhances Dex-induced attenuation of NF-κB transcriptional activity. HO-8910 cells were cotransfected with 0.2μg of NF-κB-luc alone or together with 0.8μg of blank vector or each of indicated RhoB plasmids for 8h, and then cells were incubated in medium with or without 100nM Dex for additional 24h and lysated for luciferase activity assay. Values shown represent the mean±S.E.M. of five independent experiments. *p<0.05, **p<0.01. Pathophysiology 2009 16, 267-272DOI: (10.1016/j.pathophys.2009.02.009) Copyright © 2009 Elsevier Ireland Ltd Terms and Conditions