Fig. 4

PD-L1 is a target of miR-155-5p in macrophages. A. Target Scan predicted 2 potential binding sites of miR-155-5p on PD-L1 3′-UTR. B. THP-1 cells were co-transfected with PD-L1 full-length 3’UTR plasmid and miR-cont, miR-155, miR-152 or miR-137. Luciferase activities were normalized by the Renilla control’s luciferase activity. miR-152 was set as a positive control, and miR-137 as a negative control. C. Macrophages were transfected with miRNA mimic control (miR-con) or miR-155-5p mimic (miR-155) for 48 h. PD-L1 expression was determined by immunoblotting. D-E. Macrophages pre-treated with IFN-γ (100 ng/ml, 24 h) were treated with Exo-miR-con or Exo-miR-155 at 100 μg/ml for 48 h. PD-L1 expression levels were determined by flow cytometry and immunoblotting. F-G. Macrophages pre-treated with IFN-γ (100 ng/ml, 24 h) were treated with Exo-con and Exo-NAC at 100 μg/ml for 48 h. PD-L1 expressions were detected by flow cytometry and immunoblotting. Mean Fluorescence Intensity (MFI) was statistically analyzed. H. PD-L1 expression levels in macrophages in A2780-miR-con spheroids and A2780-miR-155 spheroids as described in Fig. 4 were determined by flow cytometry. I. Macrophages were transiently transfected with PD-L1 or vector for 48 h before adding to the 3D tumor spheroids. The number of tumor infiltrating macrophages was determined by flow cytometry. Data are representative of 3 independent experiments. *p < 0.05, **p < 0.01