BTG1 knockdown in RS4;11 cells impairs GC-induced gene expression because of loss of GR expression. (A) Venn diagram summarizing microarray expression analysis in prednisolone-treated (8-hour) control RS4;11 cells and BTG1 knockdown (shRNA1) RS4;11 cells. The number of genes with a significant (2-fold change) in expression is indicated. (B) Heat map of the 200 genes with highest differential expression after prednisolone exposure in control cells. For comparison, expression changes in BTG1 shRNA 1 cells are shown in the right column. (C) mRNA expression of the GR and the GC-regulated genes SGK1 and VDR were analyzed by quantitative RT-PCR and normalized for TBP after exposure to 1 mg/mL prednisolone for up to 8 hours in RS4;11 control and BTG1 shRNA1 cells. (D) Immunoblot showing GR expression in control RS4;11 cells and in BTG1 knockdown cells in response to prednisolone treatment for up to 8 hours. (E) Immunoblot showing GR expression in RS4;11 cells transduced with a control shRNA, BTG1 shRNA1, or BTG1 shRNA1 with a GR overexpression construct. (F) MTS assay determining relative viability of RS4;11 cells transduced with a control shRNA, BTG1 shRNA1, or BTG1 shRNA1 with a GR overexpression construct after prednisolone treatment. Error bars represent the SEM of triplicate experiments. Statistical significance (P < .05) was assessed with the Student t test compared with untreated control cells.