Fig. 4

AIM2 upregulation promotes PD-L1 expression via activating STAT1 and NF-κB signaling pathways in OSCC cells. A and B RT-PCR (left)/Western blot analyses (right) for the endogenous mRNA/protein levels of PD-L1 and GAPDH (A) and histograms for the mean of fluorescent intensity (MFI) of isotype control (cyan) and PD-L1 (pink) antibodies in Flow-cytometric analysis (B) against SAS and HSC4 cells. C and D RT-PCR (upper)/Western blot analysis(middle) for the endogenous mRNA/protein levels of PD-L1 and GAPDH and histograms for the MFI of PD-L1 antibody normalized to control groups (NS and VC) in Flow-cytometric analysis against HSC4 (C) and SAS (D) cell variants. E–H Western blot analyses for phosphorylated STAT1/NF-κB, total STAT1/NF-κB and GAPDH protein levels (upper) and histograms for the DNA-binding activity of STAT1/NF-κB normalized to control groups (NS and VC) in luciferase reporter assay (lower) against HSC4 (E and G) and SAS (F and H) cell variants. I Illustration for the locations and mutated nucleotides of NF-κB binding site/IFNγ activated site in the PD-L1 promoter region subcloned into the upstream of the firefly luciferase gene. J–H Histograms for the normalized luciferase activity detected from HSC4/SAS cell variants (J), AIM2-overexpressing SAS cells (K) and HSC3/HSC4 cells (H) cells transfected with the PD-L1 promoter-driven firefly luciferase reporter vector without or with mutations at NF-κB binding site and IFNγ activated site. In A, C, D, E, F and G, GAPDH was used as an internal control of experiments. The symbol “***” and different alphabets denote p < 0.001 and 0.05, respectively