Figure 1.
The expression of PLKs in THP-1 cells.
(A) Quantitative expression of the PLK genes. THP-1 cells were cultured for 24 h in 6-well plates. The gene expression of PLKs was detected by qRT-PCR. PLK1 gene expression was designated as 100%. PLK2-4 gene expression was shown as percentage expression compared with PLK1 gene expression. (B) The effect of GW843682X on the protein expression of the PLKs. THP-1 cells were treated with 10 µM GW843682X for the indicated time periods. The protein expression of PLKs was detected by western blot. β-actin protein was detected as a loading control. (C) The effect of GW843682X on the gene expression of the PLKs. THP-1 cells were treated with 10 µM GW843682X for the indicated time periods. The gene expression of the PLKs was detected by qRT-PCR. Gene expression in the GW843682X-treated groups was shown as the fold expression relative to the non-treated groups.
Figure 2.
The effect of GW843682 (GW) on cytokine expression induced by Pam3CSK4 and LPS.
(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 TNF-α, IL-1β and IL-8 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. * P < 0.05 compared with the Pam3CSK4- or LPS-treated groups in (A-F).
Figure 3.
The effect of PLK1 RNA interference on Pam3CSK4-induced cytokine production.
(A-C) The effect of PLK1 siRNA on PLK1 expression. THP-1 cells, transfected with PLK1 siRNA as described in Materials and Methods, were untreated (A) or stimulated with 1 µg/ml Pam3CSK4 (P3C4) for 1 h (B-C). The protein expression of PLK1 was detected by western blot (A, C). The gene expression of PLK1 was detected by qRT-PCR (B). * P < 0.05 compared with the non-treated groups. (D) PLK1 RNA interference down-regulated cytokine transcript expression. THP-1 cells, transfected with PLK1 siRNA, were stimulated with 1 µg/ml Pam3CSK4 (P3C4) for 1 h. The transcript expression of TNF-α, IL-1β, and IL-8 were detected by qRT-PCR. * P < 0.05 compared with the non-treated group. (E) The effect of PLK1 siRNA on cytokine protein expression. THP-1 cells, transfected with PLK1 siRNA, were stimulated with 1 µg/ml Pam3CSK4 (P3C4) for 24 h. The protein expression of TNF-α and IL-8 was detected by ELISA. * P < 0.05 compared with the Pam3CSK4-treated alone group.
Figure 4.
The effect of Pam3CSK4 and LPS on PLK activation and expression .
(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 PLKs was detected by qRT-PCR. The PLK1 gene expression in non-treated group was designated as 1-fold. The PLK1 gene expression in Pam3CSK4- or LPS-treated groups, and the PLK2-4 gene expression in all groups was shown relative to the PLK1 gene expression in the non-treated group. * P < 0.05 compared with the control groups respectively.
Figure 5.
The effect of GW843682X (GW) on THP-1 apoptosis.
(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). * P < 0.05 compared with the non-treated groups in (C) and (D).
Figure 6.
The effect of GW843682X (GW) on the expression of TLR2, TLR4, and MyD88.
(A) The effect of GW on the gene expression of TLR2, TLR4, and MyD88. THP-1 cells were treated with 10 µM GW for the indicated time periods. The gene expression of TLR2, TLR4, and MyD88 was detected by qRT-PCR. * P < 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.
Figure 7.
GW843682X (GW) down-regulates TNF-α expression via inhibition of MAPK and NF-κB signaling.
(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. * P < 0.05 compared with the Pam3CSK4- or LPS-treated groups.