Fig. 5.

PTM inhibits IFN-γ-induced PD-L1 expression and attenuates STAT1 phosphorylation in LLCs. (a-c) LLCs were incubated with medium, and pretreated-PTM (12.5%) for 4 h and subsequently treated with IFN-γ (10 ng/mL) for 24 h. A positive sample was used for IFN-γ treatment (10 ng/mL). (a) PD-L1 expression was detected via western blot. The PD-L1 (relative to β-actin) expression was calculated using Image J. Statistical significances were estimated using a two-way analysis of variance (ANOVA); mean ± SEM; n = 3, *p < 0.05. (b) Confocal images indicate stained nuclei (blue) and mouse PD-L1 (green) being merged; scale bar, 10 µM. Mean fluorescence intensities were subsequently quantified using Zen blue; n = 20, 18, 20, and 18 single cells were used for control, PTM alone, IFN-γ, and PTM/IFN-γ, respectively; **p < 0.01, and ****p < 0.0001. n.s., not significant. (c) Flow cytometric analysis of PD-L1-PE was observed using Flowzo (n = 3). The graph indicates PD-L1 levels (clone 9A11) normalized by the lgG control. Statistical significance was determined via one-way ANOVA. Data are represented by the mean ± SEM of four representative experiments: **p < 0.01, and ***p < 0.001. (d) PTM markedly reduced IFN-γ-induced STAT1 phosphorylation at the indicated times. Cells were incubated with IFN-γ alone (positive control), and 12.5% PTM for 4 h, and subsequently treated with IFN-γ (10 ng/mL) at the indicated times; “–“, before indicated times (0, 15, 30, 60 min) indicates negative control. STAT1 phosphorylation (pSTAT1) at Tyr701, as well as at total STAT1 (relative to β-actin), was detected using western blot analysis. Line charts illustrate quantification data about pSTAT1 levels at indicated time points compared to levels induced by IFN-γ stimulation from the positive control group. Statistical differences between IFN-γ and IFN-γ/PTM were determined via a two-way ANOVA. Data are represented by the mean ± SEM of three independent experiments: ***p < 0.001 (15 min) and **p < 0.01 (30 min)