Fig. 3

RUNX1 regulates M2 polarization of TAMs. A Experimental scheme of different CMs preparation and in vitro model of cells co-culture. B THP-1 was stimulated with CMs derived from RUNX1 over-expressed (left; HCT116 vector/RUNX1^OE, RKO vector/RUNX1^OE) or knockdown (right; SW480 shNC/shRUNX1, RKO shNC/shRUNX1) CRC cells, and the IL-10 in supernatant was analyzed by ELISA. C (left) Flow cytometry analysis of macrophage polarization phenotype stimulated by CMs derived from RUNX1 over-expressed HCT116 and RKO cells. (right) Quantification of percentage of CD68⁺CD206⁺ or CD68⁺CD200R⁺ macrophages (of macrophages; n = 3). D RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 over-expressed HCT116 (left) and RKO (right) cells. (E) (left) Flow cytometry analysis of macrophage polarization state stimulated by CMs derived from RUNX1 knockdown SW480 and RKO cells. (right) Quantification of percentage of CD68⁺CD206⁺ or CD68⁺CD200R⁺ macrophages (of macrophages; n = 3). (F) RT-qPCR analysis of the expression of CD163 mRNA, CD206 mRNA, Arg1 mRNA and IL-10 mRNA in macrophages treated with CMs derived from RUNX1 knockdown SW480 (left) and RKO (right) cells. G CD206 and CD200R IF labeling of THP-1 cells treated with CRC cells-derived CMs. Data were recorded by a confocal laser scanning microscopy. Scale bars: 10 μm. H Quantification of mean fluorescence intensity of CD206 and CD200R (IntDen/Area; n = 3). I The expression of PTCH1, PTCH2, GLI1, SUFU and Shh in TAMs treated with CMs derived from RUNX1 over-expressed HCT116 cells was determined by western blotting. J (left) Flow cytometry analysis of macrophage phenotype induced by CMs derived from RUNX1 over-expressed HCT116 cells in the presence or absence of GDC-0449. (right) Quantification of percentage of CD68⁺CD206⁺ or CD68⁺CD200R⁺ macrophages (of macrophages; n = 3)