Figure 5.

MDFIC alters the transcriptional activity of GR on glucocorticoid-responsive reporter genes in A549 cells. A, A549 cells were transfected with the pMMTV-LUC reporter (left panel) or the pGRE2-LUC reporter (right panel) and either empty vector, FLAG-MDFIC, or FLAG-MDFIC(1–164). After an 18-h treatment with vehicle or 100 nm Dex, the cells were harvested, and luciferase activity was measured. Data represent the mean ± S.D. from four independent experiments performed in quadruplicate. A one-way ANOVA followed by Tukey's post hoc test was performed to determine significance. ***, p < 0.001 for Dex versus con (vehicle). ###, p < 0.001 for MDFIC Dex versus vector Dex and for MDFIC(1–164) Dex versus MDFIC Dex. B, protein lysates from A549 cells transfected as above with FLAG-MDFIC or FLAG-MDFIC(1–164) were immunoblotted with the anti-FLAG antibody (upper blot) or anti-actin antibody (lower blot). Shown is a representative immunoblot from three independent experiments. C, A549 cells were transfected with the pMMTV-LUC reporter (left panel) or the pGRE2-LUC reporter (right panel) and either NTC siRNA or MDFIC siRNA. After a 6-h treatment with vehicle or 100 nm Dex, the cells were harvested, and luciferase activity was measured. Data represent the mean ± S.D. from three independent experiments performed in quadruplicate. A one-way ANOVA followed by Tukey's post hoc test was performed to determine significance. ***, p < 0.001 for Dex versus con. ###, p < 0.001 for MDFIC siRNA Dex versus NTC siRNA Dex. D, RT-PCR analysis of MDFIC mRNA expression in A549 cells transfected as above with reporter gene and either NTC siRNA or MDFIC siRNA (Student's t test, mean ± S.E. ***, p < 0.001).