Fig. 2

(A-B) Flow cytometry results revealing membranous PD-L1 expression after Wnt pathway inhibitor treatment. HuH7 cells were treated with XAV939 (A) or IWR-1 (B) at the indicated concentration for 24 h, before 25 ng/mL IFN-γ was added for another 24 h. The cells were subsequently harvested and stained with anti-PD-L1 antibodies or isotype controls (^*p < 0.05). (C-F) Expression of CD274 mRNA after Wnt pathway inhibitor treatment. HuH7 (C-D) and Hep3B (E-F) cells were treated with XAV939 (C and E) or IWR-1 (D and F) at the indicated concentration for 24 h, before 25 ng/mL IFN-γ was added for another 24 h. The cells were subsequently harvested. CD274 mRNA expression was analyzed using RT-qPCR with GAPDH as the internal control. Expression is reported as fold changes relative to cells treated with the vehicle (^*p < 0.05). (G-H) Western blotting demonstrating the influence of the Wnt pathway inhibitor XAV939 on PD-L1 expression in cells overexpressing beta-catenin. Empty vectors or vectors overexpressing CTNNB1 (coding gene of beta-catenin) were added to the HuH7 cells after seeding. After 48 h, we treated the cells with XAV939 at the indicated concentration for 24 h before IFN-γ 25 ng/mL was added. The cells were harvested after 24 h. PD-L1 protein expression was analyzed with GAPDH as the internal control. Expression is reported as fold changes relative to cells treated with the empty vector and vehicle only (^*p < 0.05)