Fig 2: PZH suppressed the basal STAT3-IRF1 signaling to down-regulate PD-L1 expression. (A) The protein levels of p-STAT3, IRF1 and PD-L1 were dramatically attenuated in PZH treated CMT93 and HCT116 cells. (B) PZH treatment suppressed the mRNA expression of IRF1 and PD-L1 in CMT93 and HCT116 cells (n = 3 per group). The results are representative of one of three experiments. Data are presented as means ± SD, **p < 0.01, ***p < 0.001 based on one-way ANOVA.

Fig 3: PZH suppressed IFNγ stimulation signal and PD-L1 up-regulation via inhibiting the IFNGR1-JAK1-STAT3-IRF1 signaling. (A) IFNγ-induced p-JAK1, p-STAT3, IRF1, and PD-L1 protein levels were attenuated in PZH treated CMT93 and HCT116 cells, as well as the basal IFNGR1 protein level. (B) The mRNA levels of IRF1 and PD-L1 induced by IFNγ were decreased in PZH treated CMT93 and HCT116 cells (n = 3–4 per group). The results are representative of one of three experiments. Data are presented as means ± SD, ***p < 0.001 based on one-way ANOVA.

Fig 4: PZH synergistically promoted the antitumor efficacy of anti-PD-1/PD-L1 immunotherapy. (A–B) The tumor growth (A) and size (B) of CMT93 bearing mice were attenuated in the combination groups compared with the monotherapy groups at day 21 of treatment (n = 5 per group): IgG control, PZH (0.234 g/kg), PD-L1 antibody (100 μg), PZH + PD-L1 antibody combination group (PZH 0.234 g/kg and PD-L1 antibody 100 μg), PD-1 antibody (100 μg) and PZH + PD-1 antibody combination group (PZH 0.234 g/kg and PD-1 antibody 100 μg). (C) The treatment efficiency of combination group was higher than that of monotherapy with either agent (n = 5 per group). The treatment efficiency was quantified by tumor weight and normalized to the Ctrl group. (D–E) The number of tumor-infiltrating CD3+ and CD8+ cells was elevated in the combination groups with indicated treatments (n = 5 per group) as described in (A–B). CD3 and CD8 staining were performed by immunohistochemical analysis, and the number of positive cells was analyzed by Image Pro Plus 6.0 software. (F) The mRNA levels of IFNγ and Granzyme B were up-regulated in the combination groups with indicated treatments (n = 3 per group) as described in (A–B). The results are representative of one of three experiments. Data are presented as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 based on one-way ANOVA.

Fig 5: TBX2 level was correlated with programmed death ligand 1 (PD‐L1) in cutaneous squamous cell carcinoma (CSCC) specimens. (A and B) Relative PD‐L1 (CD274) mRNA (A) and protein (B) level in normal or CSCC specimens in different stages were determined by RT‐qPCR and western blot, respectively. Six normal skin specimens, 10 of CSCC‐I, 21 of CSCC‐II, 18 of CSCC‐III/IV. ns/*, the significance compared to normal specimens was analyzed by one‐way ANOVA test. (C) Pearson correlation assay of TBX2 and PD‐L1 protein level determined by western blot in CSCC specimens