Fig. 7

CXCL1^EV-dead transcriptionally increases PD-L1 expression in macrophages by activating EED signaling. A The mRNA level of PD-L1 in Raw264.7 macrophages was significantly increased by 10 ng/ml murine CXCL1 and 50 μg/ml EV-dead treatment for 24 h; (B) The promoter activity of PD-L1 in Raw264.7 cells when treated as indicated for 24 h; CXCL1-NA concentration: 5 μg/ml; (C) Diagram of the DNA-pull down-MS assay. The DEPs that bind with the PD-L1 promoter region of Raw264.7 macrophages after CXCL1 treatment for 24 h were analyzed by mass spectrometry. The TFs of PD-L1 were predicted using the hTFtarget database. EED was selected as the potential transcription factor by taking the intersection of the DEP set and the TF set. D, E The expression levels of EED and PD-L1 in Raw264.7 cells when treated as indicated for 48 h. Murine CXCL1 concentration: 10 ng/ml. Scale bar: 5 μm. F The combinational effect of CXCL1 treatment and EED knockdown on the promoter activity of PD-L1 in Raw264.7 cells when treated as indicated for 24 h; (G) The binding activity of EED with the promoter fragment of PD-L1 in Raw264.7 cells when treated as indicated for 48 h was investigated by CHIP-PCR assay. H The antisense mutation of the EED binding region in the PD-L1 promoter significantly abrogated the induction effect of CXCL1 on PD-L1 promoter activity in Raw264.7 macrophages; n = 3. Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01