Figure 3. Effect of SB203580 on steroid sensitivity and GR nuclear translocation in U937. A.

U937 cells were initially incubated with IL-2/IL-4 for 48 hours. Cells were pre-treated with SB203580 (5 µM) for 30 min followed by Dex (10⁻¹¹−10⁻⁶ M) for 1 hour and TNFα stimulation (10 ng/ml) overnight. TNFα-induced IL-8 release was evaluated by ELISA and IC₅₀dex values for Dex on IL-8 production were calculated. Values represent means of three experiments ± SEM. # p<0.05 (vs. non-treatment control; NT), and* p<0.01 (vs. treatment with IL-2/IL-4 only). B. U937 cells were incubated with IL-2/IL-4 for 48 hours. Cells were then stimulated with SB203580 (5 µM) for 30 min followed by Dex 10⁻⁶ M for 4 hours. Nuclear protein was extracted and GR was detected using SDS-PAGE/Western Blotting. TBP was detected as loading control. Ratio of GR nuclear translocation was calculated dividing GR absorbance by TBP.*** p<0.001 (IL-2/IL-4+ dex vs. treatment with IL-2/IL-4 only), # p<0.05 (IL-2/IL-4+ dex + SB vs. treatment with IL-2/IL-4+ dex), n = 3. C. U937s were stimulated with IL-2/4 for 48 hours and then stimulated with SB203580 (5 µM) for 30 minutes prior whole-cell extraction and SDS-PAGE/Western-Blotting. Phosphorylation of Serine 226 was determined with anti-S226 GR antibody normalized to GR expression. The band density was calculated by densitometry. ## p<0.01 (vs. non-treatment control),* p<0.05 (vs. treatment with IL-2/IL-4 only), n = 4.