Figure 4.

Metformin decreases the fractions of MDSCs and M2 macrophages by downregulating the mevalonate pathway for protein prenylation. (A) THP-1 cells were treated with PMA or control vehicle for six hours and then were treated with 5 mM metformin and 50 nM rapamycin for an additional 4 h. (B) Regarding the specific condition of the upstream regulator AICAR, THP-1 cells were treated with PMA and AICAR together for 4 h. Cells were harvested and examined by real-time qPCR, normalized to GAPDH expression. Data are expressed as the means ± standard errors of the two different experiments; * p < 0.05, ** p < 0.005. (C) In Western blot analysis, the expression of HMGCR was analyzed after 24 h of treatment with the control vehicle or PMA (100 μM) with or without metformin (5 mM), rapamycin (50 nM) and AICAR (0.5 mM) in THP-1 cells. (D) THP-1 cells were treated with 2 mM metformin, 10 μM YM-53601, 10 to 20 μM FTI-277 and 5 to 10 μM GGTI-298 for 48 h. Next, the expression levels of MDSC (CD33⁺/CD68⁺ and Arg-1⁺/CD68⁺) and M2 macrophage (CD206⁺/CD68⁺ and CD163⁺/CD68⁺) markers were analyzed by flow cytometry. Data are expressed as the means ± standard errors of the three different experiments; * p < 0.05, ** p < 0.005. (E) THP-1 cells were treated with PMA for 48 h with the control vehicle or co-treated with 2 mM metformin and/or 200 μM mevalonate. After 48 h, the culture supernatant was analyzed by ELISA. Date are expressed as the means ± standard errors of the four different experiments; * p < 0.05, ** p < 0.005.