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. 2019 Jul 16;10:1643. doi: 10.3389/fimmu.2019.01643

Figure 4.

Figure 4

M1 macrophages derived from BMDM upregulated expression of PD-L1 in murine HCC cells. (A) Bone marrow cells from C57BL/6 mouse were cultured with M-CSF for 7 days. Flow cytometry assay was performed to identify mature BMDM by using F4/80 and CD11b as identification markers. (B) BMDMs were stimulated by LPS or IL-4 for 12 h to generate BMDMs (LPS) or BMDMs (IL-4) with M1 or M2 phenotype, respectively. The BMDMs without stimulation were defined as M0. Expression of IL-6, TNF-α, iNOS, CD206 and Arg-1 was detected by RT-PCR. (C) Expression of IL-1β was determined by quantitative RT-PCR. (D) PD-L1 mRNA expression in the Hepa 1-6 cells treated with medium, M0S, M1S, M2S, LPS or IL-4 was quantified by RT-PCR assay. (E) PD-L1 protein expression in the Hepa 1-6 cells treated with medium, M0S, M1S, M2S, LPS, or IL-4 was quantified by flow cytometry. The experiment was carried out in triplicate and repeated at least twice. Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.