Figure 5. A20 is involved in the induction of cross-tolerance between LPS and Pam3CSK4.

(A) The effect of various PAMPs on cytokine gene expression. THP-1 cells were treated with medium, PGN (5 µg/ml), Poly (I:C) (5 µg/ml), LPS (1 µg/ml), flagellin (FGN, 0.1 µg/ml), and Pam3CSK4 (1 µg/ml) for 1 h. The gene expression of cytokines was detected by RT-PCR. β-actin gene expression was detected as loading control. (B) The effect of various PAMPs on the cytokine protein expression. THP-1 cells were treated with indicated PAMPs for 24 h. The cytokine proteins in the supernatant were detected by ELISA. * P<0.05 compared with medium group. (C) The effect of various various PAMPs on A20 protein expression. THP-1 cells were treated with indicated PAMPs for 24 h. A20 protein expression was detected by Western blot. β-actin protein expression was detected as loading control. (D) The effect of Pam3CSK4 pre-treatment on LPS-induced cytokine expression. THP-1 cells, pre-treated (Pre-T) with medium, or 1 µg/ml Pam3CSK4 for 24 h, were re-treated (Re-T) with medium, or 1 µg/ml LPS for 1 h. Cytokine gene expression was detected by qRT-PCR. * P<0.05 compared with LPS-treated alone group. (E) The effect of LPS pre-treatment on Pam3CSK4 (P3C4)-induced cytokine expression. THP-1 cells, pre-treated (Pre-T) with the indicated concentrations of LPS for 24 h, were re-treated (Re-T) with medium, or 1 µg/ml Pam3CSK4 for 1 h. Cytokine gene expression was detected by qRT-PCR. # P<0.05 compared with Pam3CSK4-treated alone group. (F) The effect of PGN, Poly(I:C) (IC), LPS, flagellin (FGN) pre-treatment on Pam3CSK4 (P3C4)-induced phosphorylation of p38 and JNK. THP-1 cells, pre-treated with medium, or 10 µg/ml PGN, or 10 µg/ml Poly(I:C)(IC), or 1 µg/ml LPS, or 100 ng/ml flagellin (FGN) for 24 h, were re-treated with medium, or 1 µg/ml Pam3CSK4 for 30 min. The phosphorylation of p38 and JNK was detected by western blot. β-actin protein was detected as loading control.