The expression of TAM differentiation and activation markers in RAW 264.7 cells was decreased by 4T1-N2 CM. (A) The mRNA and protein levels of NDRG2 in 4T1 cells were measured by RT-PCR and western blot analysis. (B) The 4T1-mock and 4T1-NDRG2 cells were cultured for 48 h, and then IL-10 and GM-CSF levels in the supernatants were determined by sandwich ELISA kits. (C) To generate TAMs, RAW 264.7 cells were treated with 4T1-M CM or 4T1-N2 CM for 24 h. Cell morphology images were captured using a micro-scope (×20). (D) The mRNA expression levels of TAM differentiation markers (MR1) and activation markers (IL-10, iNOS and ARG1) were measured by quantitative real-time PCR. (E) The CD206, PD-L1 and PD-1 expression in TAM was quantified by flow cytometry. The results are presented as the mean ± SEM. The data are representative of at least three independent experiments. *P < 0.05, **P <0.01, ***P < 0.001.
