Bone marrow cells induce PD-L1 expression on tumor cells. (A) Tumor cell surface PD-L1 expression after co-culture with BM cells for 48 hours. Cells were stained with isotype control or PE-PD-L1 antibody. PD-L1 expression level was determined by flow cytometry. Data are presented as mean ± standard error (n = 3), *P <0.05 versus B16F10 alone. Student t test (B) Intracellular PD-L1 in B16F10 cells was detected by staining with isotype control or PE-PD-L1 antibody, and PD-L1 expression level was examined using flow cytometry. Results are representative of three independent experiments. (C) Immunostaining of PD-L1 (red) expression in B16F10 cells in monoculture or co-culture with BM cells. Nucleus (blue) was stained with DRAQ5. (D) Total RNA was isolated from B16F10 cells co-cultured with BM cells and then subjected to qRT-PCR to measure the level of PD-L1. As a control, mono-cultured B16F10 cells and BM cells were separately collected using Trizol and then followed total RNA isolation to measure the level of PD-L1. The levels of GAPDH were also determined and served as an internal control for standardization. Data are presented as mean ± standard error (n = 3), *P <0.05 versus control. (E) B16F10 cells were co-cultured with BM cells for 48 hours and subjected to lysis; cell lysates were subjected to immunoblotting to detect PD-L1. β-actin was used as a loading control. MFI = Median Fluorescence Intensity, BM = Bone Marrow.