Figure 3.

The silencing of GRβ affected the effect of DHEA on protein expression and cytokine production. THP-1 cells were transfected for 48 h with siRNA for the GRβ isoform. Transfection was carried out using Lipofectamine 2000 as detailed in Methods. After 48 h cells were treated for 16 h with DHEA (10 and 100 nM) or DMSO as a vehicle control. Cellular extracts were examined by Western blot using specific antibodies, respectively, against GRβ isoform (A), GRα (B) and RACK1 protein (C). β-Actin expression was detected to normalize the samples. The images are representative of Western blot results. After DHEA treatment, cells were exposed to LPS (0.1 μg·mL⁻¹) and IL-8 release was evaluated 24 h later in culture supernatants by elisa (D). Each value in the graph represents the mean ± SD of three independent experiments. Statistical analysis was performed with Tukey's multiple comparison test with **P < 0. 01, ***P < 0. 001 versus silenced control (GRβ siRNA) and with #P < 0. 05 versus no silenced control.