Fig. 5: Tumor supernatants of NPC upregulated PD-L1 expression on HUVECs via crosstalk between NF-κB and STAT3 signaling pathways.

A, B GSEA results indicated that the JAK-STAT and NF-κB pathways were significantly upregulated. C HUVECs were pretreated with or without tumor CM of NPC. The expression of phospho-P65, total P65, phospho-STAT3, total STAT3, and PD-L1 was determined by WB. D HUVECs were pretreated with or without NF-κB or STAT3 signaling pathway inhibitors for 1 h, followed by co-culture with or without tumor CM of NPC. The expression of phospho-P65, total P65, phospho-STAT3, total STAT3, and PD-L1 was determined by WB. E HUVECs were pretreated with or without NF-κB or STAT3 signaling pathway inhibitors for 1 h, followed by co-culture with or without tumor CM of NPC. WB detection of P65 and STAT3 in the nuclear and cytoplasm, respectively. F Immunofluorescence assay to determine the effect of STAT3 pathway inhibitor (STATi, 10 μM) and NF-κB pathway inhibitor (NF-κBi, 10 μM) and supernatants of NPC tumor cell (H-E, C666-1) on P65 nuclear translocation in HUVECs. (Left) representative immunofluorescence images of P65 staining. Magnification: 1000×. (Right) The mean density values of P65 levels in the cytoplasm and nucleus of HUVECs in each group. Data are mean ± SD, two-tailed t-test. G Immunofluorescence assay to determine the effect of STAT3 pathway inhibitor (STATi, 10 μM) and NF-κB pathway inhibitor (NF-κBi, 10 μM) and supernatants of NPC tumor cell (H–E, C666-1) on STAT3 nuclear translocation in HUVECs. (Left) representative immunofluorescence images of STAT3 staining. Magnification: 1000×. (Right) The mean density values of STAT3 levels in the cytoplasm and nucleus of HUVECs in each group. Data are mean ± SD, two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.