Fig. 4

CXCL1^EV-dead induces macrophage M2 polarization by activating PD-L1 expression. A Chemokine array assay was conducted to characterize the differences in chemokine content between EV-dead and EV-alive. An ELISA assay was conducted to compare the relative CXCL1 content in EV-dead and EV-alive. B Changes in M2 phenotype polarization of Raw264.7 macrophages when treated with 10 ng/ml murine CXCL1, 50 μg/ml EV-dead, 50 μg/ml EV-dead^shCXCL1, 5 μg/ml CXCL1 neutralizing antibody (NA), or EV-dead and CXCL1-NA combination for 48 h. C Representative images of Transwell assay. Raw264.7 macrophages were treated as indicated for 48 h and then co-cultured with 4 T1 cells. Scale bar: 200 μm. D–F Expression changes of CXCL1 and PD-L1 in Raw264.7 macrophages when treated as indicated for 48 h. Scale bar: 10 μm. G The results of the flow cytometry assay suggested that 50 μg/ml EV-dead treatment for 48 h induced the M2 polarization of Raw264.7 macrophages by activating PD-L1 expression; n = 3. Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01