Figure 4

Induction of PD-L1 by BM cells is dependent on the p38 signaling pathway. (A) B16F10 cells co-cultured with BM cells were stained with pERK, pJNK, pp38, pAKT, pmTOR, pp70-S6K, pSTAT1, pSTAT3 pSTAT4, and pSTAT5 antibodies and analyzed using flow cytometry. Fold increase represents the MFI ratio between co-culture and monoculture (MFI of B16F10 in co-culture/MFI of B16F10 in monoculture). (B) B16F10 cells co-cultured with BM cells were subjected to lysis, and cell lysates were subjected to immunoblotting to detect PD-L1 and p-p38 levels. β-actin was used as a loading control. (C) PD-L1 expression was determined in B16F10 cells co-cultured with BM cells and p38 inhibitor PH797804 by staining with PD-L1 antibody and flow cytometry analysis. (D) B16F10 cells were treated with 1 μM PH797804 during co-culture with BM cells for 48 hours. Cells were subjected to lysis, and cell lysates were subjected to immunoblotting to detect PD-L1. β-actin was used as a loading control. (E) B16F10 cells were treated with 5 μM PH797804 during monoculture or co-culture with BM cells for 48 hours and then stained with annexin V and PI to determine cell viability by flow cytometry. Data are presented as mean ± standard error (n = 3). *P <0.05 versus B16F10 alone, student t test. MFI = Median Fluorescence Intensity, BM = Bone Marrow.