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. 2020 Aug 24;43(9):265–272. doi: 10.1097/CJI.0000000000000335

FIGURE 5.

FIGURE 5

TNF-α enhances Tc9-cell differentiation through STAT5 and NF-κB. A, Naive CD8+ T cells were cultured under Tc9-polarizing conditions with or without the addition of TNF-α for 3 hours. Western blots examined the protein levels of the phosphorylated STAT5 (pSTAT5), IKKα/β (p-IKKα/β), and phosphorylated STAT6 (pSTAT6) in T cells. β-actin was used as a loading control. B, Naive CD8+ T cells were cultured under Tc9-polarizing conditions with or without the addition of TNF-α for 6 hours. Western blots examined the protein levels of the p65 and p50 in the nucleus of T cells. Histone3 was used as a loading control. C–F, Naive CD8+ T cells were cultured under Tc9-polarizing conditions in the presence or absence of TNF-α with or without (DMSO) the addition of STAT5 inhibitor (STAT5i) or NF-κB inhibitors [bortezomib (Bor), CAS 213546-53-3 (CAS), and JSH-23] for 2 days. C and E, Quantitative polymerase chain reaction analysis of Il9 expression in T cells. D and F, Flow cytometry analysis of IL-9-expressing CD8+ (IL-9+CD8+) T cells. Numbers in the dot plots represent the percentages of IL-9+CD8+ T cells. Right, summarized results of 3 independent experiments obtained as at left. Data are representative of 3 (A, B, D, F) independent experiments or presented as mean±SD of 3 (C–F) independent experiments. *P<0.05; **P<0.01. DMSO indicates dimethyl sulfoxide; IL, interleukin; NF-κB, nuclear factor-κB; TNF, tumor necrosis factor.