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. 2023 Jan 31;11(1):e003837. doi: 10.1136/jitc-2021-003837

Figure 5.

Figure 5

tmTNF-α as a ligand increases the expression of PD-L1. MDA-MB-468 cells as target cells were cocultured with 100 ng/mL sTNF-α or tmTNF-α stably transfected, fixed 293 T cells at an E:T ratio of 10:1. A neutralizing antibody against human TNF-α was added to fixed tmTNF-α-expressing 293 T cells for 30 min prior to addition to target cells. After stimulation for 24 hours, PD-L1 expression was detected by flow cytometry (A) and western blot (B), and their quantitative data. (C) Representative images of western blot for the degradation of IκΒα and the phosphorylation of the p65, p38 and AKT in MDA-MB-468 cells after stimulation with both forms of TNF-α for 30 min. (D, E) MDA-MB-468 cells were pretreated with or without a p38 inhibitor SB203580 (0.6 M) for 1 hour, followed by stimulation with both forms of TNF-α for 30 min (for signal pathways) or 24 hours (for PD-L1 expression). Representative images of western blot for the p38 phosphorylation (D) and the PD-L1 expression (E) from three independent experiments and quantitative data. All quantitative data represent means±SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus control. E:T, effector:target; sTNF-α, secreted tumor necrosis factor alpha; tmTNF-α, transmembrane tumor necrosis factor alpha; TNF-α, tumor necrosis factor alpha.