Figure 1.
Induction of differentiation of THP-1 and HL60 cells by combined treatment with retinoid and 1,25(OH)2D3.
(A) NBT-reducing activities. Cells were treated with vehicle control (Cont), 100 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH)2D3 (D3) for 5 days. *, p<0.05; **, p<0.01; ***, p<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). (B) Morphological changes of THP-1 and HL60 cells treated with 9cRA and/or 1,25(OH)2D3. Cells were treated with vehicle control (Cont), 100 nM 9cRA and/or 100 nM 1,25(OH)2D3 for 5 days and the cell smears were stained with May-Grünwald-Giemsa. (C) Cell proliferations. Cells (1×105/ml) were cultured with vehicle control (Cont), 100 nM 9cRA and/or 100 nm 1,25(OH)2D3 (D3), and cell numbers were counted at indicated days. *, p<0.05; **, p<0.01; ***, p<0.001 vs Cont; ###, p<0.001 vs 9cRA; +++, p<0.001 vs D3 (one-way ANOVA followed by Tukey’s multiple comparisons). †††, p<0.001 (two-way ANOVA).
Figure 2.
Effects of combined treatment with retinoid and 1,25(OH)2D3 on cell surface CD14 and CD11b expression and CD14 mRNA expression.
Representative histograms of CD14 expression (A) and CD11b expression (B) in THP-1 cells. Cells were treated with vehicle control (Cont), 100 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH)2D3 (D3) for 96 hours. Filled curves, vehicle control. Similar results were obtained from repeated experiments. (C) CD14 mRNA levels in THP-1 and HL60 cells. Cells were treated with vehicle control (Cont), 30 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH)2D3 (D3) for 72 hours. **, p<0.01; ***, p<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).
Figure 3.
Expression of marker genes of M1 and M2 macrophages.
mRNA levels of the M2 markers CD163 (A), ARG1 (B), IL10 (C), and the M1 marker IL12B (D) in THP-1 and HL60 cells. (E) mRNA levels of the M2 marker TGFB1, the M1 markers TNF, IL6, and NOS2 in THP-1 cells. Cells were treated with vehicle control (Cont), 30 nM 9cRA, or ATRA in the absence or presence of 100 nM 1,25(OH)2D3 (D3) for 72 hours. *, p<0.05; **, p<0.01; ***, p<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). n.d., not detected.
Figure 4.
Cell surface expression of CD163 in THP-1 cells.
(A) Representative histograms of CD14 and CD163 expression. (B) Quantification of mean fluorescence intensity of CD163 and CD14 expression. (C) Quantification of percentages of CD163+/CD14+ cells and CD163−/CD14+ cells. Cells were treated with vehicle control (Cont), 100 nM 9cRA and/or 100 nM 1,25(OH)2D3 (D3) for 96 hours. **, p<0.01; ***, p<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).
Figure 5.
Secreted IL-10 production in THP-1 and HL60 cells.
Cells were treated with vehicle control (Cont), 30 nM 9cRA and/or 100 nM 1,25(OH)2D3 (D3) for 72 hours and secreted IL-10 levels in media were measured. **, p<0.01; ***, p<0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). n.d., not detected.