The effect of Pam3CSK4 and LPS on PLK activation and expression .

<p>(A, B) Pam3CSK4 and LPS induce dose-dependent activation of PLK1. THP-1 cells were treated with the indicated concentrations of Pam3CSK4 (A) or LPS (B) for 30 min. The phosphorylation of PLK1 was detected by western blot. β-actin protein was detected as loading control. ‘+’ indicated the non-specific bands. (C, D) Pam3CSK4 and LPS induced time-dependent activation of PLK1. The THP-1 cells were treated with 1 µg/ml Pam3CSK4 (C) or LPS (D) for the indicated time periods. The phosphorylation of PLK1 was detected by western blot. β-actin protein was detected as loading control. ‘+’ indicated the non-specific bands. (E) The effect of Pam3CSK4 and LPS on PLK gene expression. THP-1 cells were treated with 1 µg/ml Pam3CSK4 or LPS for 24 h. The gene expression of <i>PLKs</i> was detected by qRT-PCR. The <i>PLK1</i> gene expression in non-treated group was designated as 1-fold. The <i>PLK1</i> gene expression in Pam3CSK4- or LPS-treated groups, and the <i>PLK2-4</i> gene expression in all groups was shown relative to the <i>PLK1</i> gene expression in the non-treated group. * <i>P</i> < 0.05 compared with the control groups respectively.</p

The effect of Pam3CSK4 on the expression of TLR1, 2, MyD88, and the negative regulatory molecules.

<p>(A) TLR1, 2, and MyD88 gene expression. THP-1 cells were treated with the indicated concentrations of Pam3cks4 for 24 h. The gene expression of TLR1, 2, and MyD88 was detected by RT-PCR. β-actin gene expression was detected as loading controls. (B) IRAK-M, ST2, SOCS1, SIGIRR gene expression. (C, D) A20 gene expression. THP-1 cells were treated with the indicated concentrations of Pam3CSK4 for 24 h (C) or with 1 µg/ml Pam3CSK4 for the indicated time periods (D). A20 gene expression was detected by RT-PCR. β-actin gene expression was detected as loading controls. (E, F) A20 protein expression. THP-1 cells were treated with the indicated concentrations of Pam3CSK4 for 24 h (E) or with 1 µg/ml Pam3CSK4 for the indicated time periods (F). A20 proteins were detected by western blot. β-actin proteins were detected as loading controls.</p

GW843682X (GW) down-regulates TNF-α expression via inhibition of MAPK and NF-κB signaling.

<p>(A, B) The effect of GW on Pam3CSK4- and LPS-induced signal transduction. THP-1 cells, starved overnight and pre-treated with the indicated concentrations of GW for 30 min, were re-stimulated with 1 µg/ml Pam3CSK4 (P3C4) (A) or LPS (B) for 30 min. The phosphorylation of ERK, JNK, p38, and NF-κB p65 was detected by western blot. β-actin protein expression was detected as loading control. (C, D) The effect of GW on Pam3CSK4- and LPS-induced NF-κB p65 nuclear localization. THP-1 cells were treated as in (A) (C) and (B) (D). Total proteins in the nucleus were extracted, and NF-κB p65 protein was detected by western blot. Histone H3 protein expression was detected as a loading control. (E, F) Pam3CSK4 and LPS induced TNF-α via the MAPK and NF-κB pathways. THP-1 cells, pre-treated with 10 µM U0126 (U), or SP600125 (SP), or SB203580 (SB), or Bay11-3072 (BAY) for 30 min, were re-stimulated with 1 µg/ml Pam3CSK4 (P3C4) (E) or LPS (F) for 24 h. TNF-α secreted in the supernatant was detected by ELISA. * <i>P</i> < 0.05 compared with the Pam3CSK4- or LPS-treated groups. </p

The pre-treatment with Pam3CSK4 down-regulates the activation of MAPKs and NK-κB induced by the re-treatment with Pam3CSK4.

<p>(A, B) MAPK phosphorylation. THP-1 cells, pre-treated with 1 µg/ml Pam3CSK4 for 6 h, were washed with PBS twice, and cultured with flesh medium for 18 h, then were re-treated with 100 ng/ml Pam3CSK4 for the indicated time periods (A) or with the indicated concentrations of Pam3CSK4 for 30 min (B). The phosphorylation of MAPKs was detected by western blot. ERK proteins were detected as loading controls. (C, D) IKK-α/β, IκB-α activation. THP-1 cells were treated as described as (A) (C), or (B) (D). The expression of phosphorylated IKK-α/β, and the total IκB-α were detected by western blot. β-actin proteins were detected as loading controls.</p

The effect of GW843682X (GW) on THP-1 apoptosis.

<p>(A) GW induced dose-dependent cell death. THP-1 cells were treated with the indicated concentrations of GW for 24 h. Unfixed cells were stained with FITC-annexinV/PI. Cell apoptosis was measured by flow cytometry. (B) GW induced time-dependent cell death. THP-1 cells were treated with 10 μM GW for the indicated time periods. Cell apoptosis was measured as described in (A). (C) The quantitative data for (A). (D) The quantitative data for (B). * <i>P</i> < 0.05 compared with the non-treated groups in (C) and (D).</p

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

<p>(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. * <i>P</i><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. * <i>P</i><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. # <i>P</i><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.</p

A20 is responsible for the induction of tolerance.

<p>(A) Western blot analysis of A20 expression in scramble siRNA-, or A20 siRNA-transfected THP-1 cells, pre-treated (Pre-T) with medium, or 1 µg/ml Pam3CSK4 (P3C4) for 24 h, and re-treated (Re-T) with 1 µg/ml Pam3CSK4 for 60 min. (B) Real-time RT-PCR analysis of IL-1β, and IL-8 gene expression in scramble siRNA, or A20 siRNA-transfected THP-1 cells, treated as described as (A). * <i>P</i><0.05 compared with no pre-treatment groups. (C) Western blot analysis of p38, and JNK phosphorylation in scramble siRNA-, or A20 siRNA-transfected THP-1 cells, pre-treated with medium, or 1 µg/ml Pam3CSK4 for 24 h, and re-treated (Re-T) with 1 µg/ml Pam3CSK4 for 30 min. (D) Real-time RT-PCR analysis of A20 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells. (E) RT-PCR analysis of TNF-α, IL-1β, and IL-8 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells, treated with medium, or 1 µg/ml Pam3CSK4, or 1 µg/ml LPS for 1 h. (F) Real-time RT-PCR analysis of TNF-α, and IL-8 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells, treated as described as (E). * <i>P</i><0.05 compared with the Mock-transfected groups.</p

Pre-treatment (Pre-T) with Pam3CSK4 suppresses the pro-inflammatory cytokines induced by Pam3CSK4 re-treatment (Re-T).

<p>(A) Cytokine gene expression. THP-1 cells, pre-treated with the indicated concentrations of Pam3CSK4 for 6 h, were washed with PBS twice, and cultured in flesh medium for 18 h, then were re-treated with 1 µg/ml Pam3CSK4 for 1 h. The gene expression of pro-inflammatory cytokines was detected by RT-PCR. β-actin gene expression was detected as loading controls. (B) Quantitative real-time RT-PCR analysis of cytokine gene expression. THP-1 cells were treated as described as (A). The gene expression of cytokines was detected by qRT-PCR. * <i>P</i><0.05 compared with the non-pre-treated groups. (C) ELISA analysis of cytokine protein expression. THP-1 cells, pre-treated with the indicated concentrations of Pam3CSK4 for 6 h, were washed with PBS twice, and cultured in flesh medium for 18 h, then were re-treated with 1 µg/ml Pam3CSK4 for 24 h. Cytokine proteins in the supernatant were detected by ELISA. * <i>P</i><0.05 compared with the non-pre-treated groups.</p

The effect of GW843682X (GW) on the expression of TLR2, TLR4, and MyD88.

<p>(A) The effect of GW on the gene expression of <i>TLR2, TLR4</i>, and <i>MyD88</i>. THP-1 cells were treated with 10 µM GW for the indicated time periods. The gene expression of <i>TLR2</i>, <i>TLR4</i>, and <i>MyD88</i> was detected by qRT-PCR. * <i>P</i> < 0.05 compared with the non-treated group. (B) The effect of GW on the protein expression of TLR2 and TLR4. THP-1 cells were treated with 10 µM GW for 24 h. TLR2 and TLR4 protein expression was detected by FACS. (C) The effect of GW on the protein expression of MyD88. THP-1 cells were treated with 10 µM GW for the indicated time periods. The protein expression of MyD88 was detected by western blot. β-actin protein expression was detected as loading control.</p

The effect of GW843682 (GW) on cytokine expression induced by Pam3CSK4 and LPS.

<p>(A, B) Gene expression in THP-1 cells. The cells, pre-treated with the indicated concentrations of GW for 30 min, were re-stimulated with 1 µg/ml Pam3CSK4 (P3C4) (A) or 1 µg/ml LPS (B) for 1 h. The gene expression of <i>TNF-α</i>, <i>IL-1β</i> and <i>IL-8</i> was detected by qRT-PCR. (C, D) Protein expression in THP-1. The cells pre-treated with the indicated concentrations of GW for 30 min were re-stimulated with 1 µg/ml Pam3CSK4 (P3C4) (C) or LPS (D) for 24 h. The cytokine proteins secreted in the supernatant were detected by ELISA. (E, F) The protein expression of cytokines in primary mouse peritoneal macrophages. Mouse peritoneal macrophages were prepared as described in Materials and Methods and treated as described in (C) (E) or (D) (F). The cytokine proteins secreted in the supernatant were detected by ELISA. * <i>P</i> < 0.05 compared with the Pam3CSK4- or LPS-treated groups in (A-F).</p