Fig. 5

Apatinib downregulated VEGFR2/STAT3/PD-L1 pathway in NSCLC cells and reduced the immunosuppressive TME. a Western blot analysis of VEGFR2 and p-VEGFR2 (Tyr1175) expression and qRT-PCR analysis of VEGFR2 expression in A549 and H1299 cells after aptinib treatment for 48 h. The data are presented as mean ± SD (n = 3). **p < 0.01 vs. control. b and c Western blot analysis of p-STAT3, STAT3, c-Myc and PD-L1 expressions in A549 and H1299 cells after indicated treatment. d Immunofluorescence staining of p-STAT3 in A549 and H1299 cells after apatinib treatment for 4 h (p-STAT3: green; DAPI: blue). Scar bar = 50 μm. e Western blot analysis of p-STAT3, STAT3 and PD-L1 expressions in A549 and H1299 cells after apatinib treatment with or without pretreatment of IL-6 for 48 h. f Western blot and qRT-PCR analysis of PD-L1 expression in THP-1-derived macrophages with or without stimulation with CM from A549 and H1299 cells. The data are presented as mean ± SD (n = 3). *p <0.05, **p < 0.01 vs. A549 or H1299-CM control. ^#p < 0.05, ^#p < 0.01 vs. medium control. g and h Jurkat cells were activated by stimulation with anti CD3/CD28 antibodies and then co-cultured with non-treated or apatinib (10 μM) pretreated A549 or H1299 cells; the Jurkat cells were collected for CD69 detection by flow cytometry (g) and the co-culture medium was collected for IFN-γ secretion by ELISA assay (h). The data are presented as mean ± SD (n = 3). ^&& p < 0.01 vs (−) Jurkat cell only group. **p < 0.01 vs. A549 + Jurkat or H1299 + Jurkat control group. ^## p < 0.01 vs. Jurkat cells only stimulated with anti CD3/CD28 antibodies group