Fig. 2

HBV-DNA-Pol upregulates PD-L1 expression at the transcriptional level. A, B PD-L1 was identified as a differential immune checkpoint molecule. A RNA extracts from HBV-DNA-Pol⁺ Huh7 cells or control cells were harvested and analyzed using qRT-PCR to determine the levels of relevant immune checkpoint molecules. GAPDH mRNAs was used as a control. Three independent experiments were performed for all statistical comparisons (mean ± S.D., n = 3, Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant. B Heat map based on the RT-qPCR results. C–E HBV-DNA-Pol increases PD-L1 protein levels. C Extracts from HBV-DNA-Pol⁺ Huh7 cells or control cells were collected and analyzed by western blot analysis with anti-PD-L1 and anti-GAPDH antibodies. D HBV-DNA-Pol⁺ Huh7 cells or control cells were subjected to immunofluorescence staining with an antibody against PD-L1 and stained with DAPI to visualize the nuclei. Images were digitally merged. Scale bar, 10 μm. E Cell-surface PD-L1 was measured by flow cytometry in HBV-DNA-Pol⁺ Huh7 or control cells. The results are plotted after quantitation (F). Three independent experiments were performed for all statistical comparisons (mean ± S.D., n = 3, Student’s t-test). ***P < 0.001. G HBV-DNA-Pol does not affect PD-L1 mRNA stability. HBV-DNA-Pol⁺ Huh7 cells or control cells were treated with 1 μg/ml Actinomycin D (ActD) for the indicated periods and then harvested for PD-L1 mRNA detection. H HBV-DNA-Pol does not affect the stability of PD-L1. HBV-DNA-Pol⁺ Huh7 cells or control cells were treated with 500 μg/ml cycloheximide (CHX) for the indicated periods and then harvested for immunoblotting with anti-PD-L1 and anti-GAPDH antibodies (left). The results are plotted after quantification (right)