Fig 1.
Effects of dexamethasone (Dex) on CCL2 expression and monocytic cell migration.
(A) Levels of CCL2 transcript were assessed by real-time PCR. The y-axis values represent increases in CCL2 mRNA levels normalized to GAPDH levels, relative to that of the non-treated THP-1 cells (control). Data are expressed as the means ± SD (n = 3 replicates for each group). (B) Culture media were isolated, and the levels of CCL2 protein in the media were measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). (C) Monocytic cells were exposed to the conditioned media isolated above, and migration of monocytic cells was measured by chemotaxis assay. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ** P < 0.01 vs. control; ### P < 0.001 vs. 27OHChol; ## P < 0.01 vs. 27OHChol.
Fig 2.
Effects of dexamethasone (Dex) on superproduction of CCL2.
(A) Levels of CCL2 transcript were assessed by real-time PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). (B) Culture media were isolated, and the amount of CCL2 protein secreted into the media was measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ** P < 0.01 vs. control; ### P < 0.001 vs. 27OHChol; +++ P < 0.001 vs. 27OHChol plus LPS.
Fig 3.
Attenuated production of CD14 protein by dexamethasone (Dex).
(A) CD14 on surface of THP-1 cells was labeled with fluorescein isothiocyanate (FITC), and the fluorescence was analyzed by flow cytometry. The x axis represents relative fluorescence intensity of the labeled cells, and the y axis represents the number of cells found at each fluorescence level. (B) Cell extracts were obtained from THP-1 cells after treatment with or without 27OHChol and dexamethasone, followed by Western blot analysis to detect CD14 and β-actin. Data are representative of three independent experiments. (C) Culture media were isolated, and the amount of CD14 protein secreted into the media was measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ### P < 0.001 vs. 27OHChol.
Fig 4.
Effects of dexamethasone (Dex) on expression of MMP-9 induced by 27OHChol.
(A) Levels of MMP-9 transcript were assessed by real-time PCR. Data are expressed as the means ± SD (n = 3 replicates for each group). (B) Culture media were isolated, and the levels of MMP-9 in the media were measured by ELISA. Data are expressed as the means ± SD (n = 3 replicates for each group). The results shown are the representative of three independent experiments. *** P < 0.001 vs. control; ### P < 0.001 vs. 27OHChol; # P < 0.05 vs. 27OHChol. (C) The activity of MMP-9 secreted by cells was assessed by gelatin zymography. Control THP-1 cells were cultured for 48 h in the medium alone. Data are representative of three independent experiments.
Fig 5.
Effects of dexamethasone on p65 subunit expression.
Serum-starved THP-1 cells (2.5 × 105 cells/ml) were cultured for the indicated time periods with 27OHChol (6.2 μM) in the absence or presence of 1 μM dexamethasone (Dex). (A) Levels of p65 transcript were assessed by real-time PCR. The results shown are the representative of three independent experiments. Data are expressed as the means ± SD (n = 3 replicates for each group). Statistical differences among treatment time periods were evaluated by One-way ANOVA, as descried in Materials and Methods. ** P < 0.01 vs. 0 h of 27OHChol; * P < 0.05 vs. 0 h of 27OHChol. Statistical differences between treatment with 27OHChol and 27OHChol+Dex at the indicated time periods were evaluated by the Student’s paired t-test. ## P < 0.01 vs. 27OHChol+Dex at 6 h; # P < 0.05 vs. 27OHChol+Dex at 9 h. (B) Whole cell extracts were isolated from the cells and subjected to immunoblotting for p65, phosphorylated p65 and β-actin. Data are representative of three independent experiments.