The effect of GSK3 inhibition on Pam3CSK4 (P3C4)-induced cytokine expression.

<p>(A) The effect of NF-κB inhibition on the induction of A20, TNF-α and IL-1β. THP-1 cells, pre-treated with the indicated concentrations of Bay11072 for 30 min, were re-stimulated with or without 100 ng/ml Pam3CSK4 (P3C4) for 1 h. The induction of A20, TNF-α and IL-1β was detected by qRT-PCR. * <i>P</i><0.05 compared with the Pam3CSK4-treated alone groups. (B) The effect of Pam3CSK4 on the expression of GSK3-α and β. THP-1 cells were treated with 1 µg/ml Pam3CSK4 for the indicated time periods. The expression of GSK3-α and β was detected by western blot. β-actin protein was detected as loading control. (C) GSK3 inhibitors down-regulated Pam3CSK4-induced A20 protein expression. THP-1 cells, pre-treated with the indicated concentration of SB216763 (SB) for 1 h, were re-treated with 1 µg/ml Pam3CSK4 (P3C4) for 6 h. The protein expression of A20 was detected by western blot. β-actin protein was detected as loading controls. (D) GSK3 inhibitors down-regulated Pam3CSK4-induced A20 gene expression. THP-1 cells, pre-treated with the indicated concentration of SB for 1 h, were re-treated with 1 µg/ml Pam3CSK4 (P3C4) for 1 h. The gene expression of A20 was detected by qRT-PCR. * <i>P</i><0.05 compared with Pam3cks4-treated alone group. (E) GSK3 inhibitors up-regulated Pam3cks4-induced cytokines. THP-1 cells, pre-treated with the indicated concentrations of SB216763 (SB) for 1 h, were re-treated with 1 µg/ml Pam3CSK4 (P3C4) for 24 h. The cytokines secreted in the supernatant were detected by ELISA. * <i>P</i><0.05 compared with Pam3cks4-treated alone group. (F and G) GSK3 inhibition reversed Pam3CSK4 pre-treatment-induced tolerance. THP-1 cells, treated with SB216763 for 1 h, were pre-treated (Pre-T) with Pam3CSK4 (0.1 µg/ml) for 24 h, then were re-treated (Re-T) with Pam3CSK4 (0.1 µg/ml) for 1 h (F) or 24 h (G). The cytokine gene and protein expression were detected by RT-PCR (F) or ELISA (G) respectively. * <i>P</i><0.05 compared with SB216763-non-treated but Pam3CSK4-pre-, and re-treated groups.