Fig 1: Casp8 downregulates PD-L1 protein expression. A, HepG2 cells were transfected with siNC or siCasp8 for 24 h and stimulated with IFN-? (50 ng/mL) for 24 h. Induced PD-L1 expression on the cytomembrane was quantified by flow cytometry. The experiment was carried out in triplicate and repeated at least twice. The data show the means ± SD, P-value<.01 (**). B, Western blot analysis of IFN-?–induced PD-L1 expression in the HepG2 cells transfected with Casp8 plasmids for 48 h. C, Western blot analysis of PD-L1 expression in 293T cells expressing His-PD-L1 and transfected with Casp8 plasmids or siCasp8. Exogenous His-PD-L1 in cells was detected by the anti-6xHis antibody. D, Western blot analysis of PD-L1 expression in 293T cells expressing His-PD-L1 and transfected with Casp8 plasmids at different concentration gradients (0.5 µg, 1 µg, 1.5 µg, and 2 µg). E, F, Western blot analysis of the expression of intrinsic PD-L1 in A375 and MDA-MB-231 cells after transfection with Casp8 plasmids (E) or siCasp8 (F) for 24 h or 48 h. G, Hep1-6 and B16 cells were infected with shNC or shCasp8, and the expression of PD-L1 and Casp8 was detected by Western blot analysis. H, Confocal microscopy image showing the protein expression of PD-L1 and Casp8 in MDA-MB231 and A375 cells (indicated by white arrows). Scale bar: 10 µm for the MDA-MB231 cells and 5 µm for the A375 cells
Fig 2: A20 facilitates the ubiquitination of the PD-L1 protein. A, MDA-MB231 and A375 cells were transfected with A20 plasmids with different concentration gradients (0.5 µg, 1 µg, 1.5 µg, and 2 µg) for 48 h, and PD-L1 expression was measured by Western blot analysis. B, Western blot analysis of PD-L1 expression in A375 cells transfected with A20 plasmids in the presence of MG132 (3 µmol/L) for 48 h. C, A20-WT, the ZnF4 mutant, and the ZnF7 mutant were transfected into MDA-MB231 and A375 cells, respectively. Western blot analysis was performed to measure the expression of PD-L1 48 h after transfection. D, Analysis of ubiquitin PD-L1 in MDA-MB231 and A375 cells, which were transfected with A20-WT, the ZnF4 mutant, or the ZnF7 mutant for 24 h, followed by treatment with MG132 (3 µmol/L) for 24 h
Fig 3: Activated Casp8 induces ubiquitination of the PD-L1 protein. A, 293T cells expressing His-PD-L1 were transfected with vector or Casp8 plasmids in the presence of Z-IETD-FMK (50 µmol/L) or Z-VAD-FMK (10 µmol/L) for 24 h, and exogenous His-PD-L1 in the cells was detected by the anti-6xHis antibody. B, Western blot analysis of PD-L1 expression in 293T cells expressing His-PD-L1 and transfected with pcDNA3.1, Casp8-WT, or Casp8-C360S mutation. C, Western blot analysis of His-PD-L1 expression in 293T cells expressing His-PD-L1, transfected with Flag-Casp8, and treated with MG132 (3 µmol/L) for 24 h. D, Protein stability of His-PD-L1 in 293T cells. The cells were transfected with pcDNA3.1, Casp8-WT, or Casp8-C360S and treated with cycloheximide (25 µg/mL). Western blot analysis of His-PD-L1 expression at the indicated times. E, Ubiquitination assay of His-PD-L1 in 293T cells. The cells were transfected with various plasmids as described and treated with DMSO or MG132 (3 µmol/L) for 24 h. Ubiquitinated PD-L1 was pulled down by anti-6xHis tag antibody, and Western blotting was performed with anti-ubiquitin antibody. F, 293T cells were transfected with the indicated plasmids of different concentration gradients and treated with MG132 (3 µmol/L). Ubiquitinated PD-L1 was immunoprecipitated by anti-6xHis antibody and detected by Western blot assay
Fig 4: A20 is required for Casp8-induced PD-L1 degradation. A, Confocal microscopy image showing the protein expression of PD-L1 and A20 in MDA-MB231 and A375 cells. Scale bar: 10 µm for the MDA-MB231 cells and 5 µm for the A375 cells. B, Immunoprecipitation assay analysis of the interaction of PD-L1 and A20 or Casp8. The cells were cotransfected with PD-L1-GFP and Casp8-Flag plasmids, and exogenous Casp8 and endogenous A20 were immunoprecipitated with anti-GFP antibody. C-E, Western blot analysis of the expression of PD-L1 in MDA-MB231 (C), A375 (D), and B16 cells (E) after cotransfection with siA20 and Casp8 plasmids for 48 h. ImageJ was used to analyze the gray of lanes. Histogram indicated the intensity of PD-L1/GAPDH. P <.05 (*), unpaired two-tailed t-test. F-H, Ubiquitination assays of PD-L1 in MDA-MB231 (F), A375 (G), and B16 (H) cells transfected with siA20 and Casp8 plasmids. Ubiquitin PD-L1 was immunoprecipitated with an anti-PD-L1 antibody and subjected to Western blotting with an anti-ubiquitin antibody
Fig 5: Knocking down Casp8 suppresses tumor immunogenicity by upregulating PD-L1. A-D, Nude mice (A, B) (n = 7 per group) were injected subcutaneously with B16/shNC or B16/shCasp8 cells (1.5*105); C57BL/6J mice (C, D) (n = 6 per group) were injected subcutaneously with B16/shNC or B16/shCasp8 cells (2 * 105). Tumor volume (A, C) and tumor weight (B, D) were measured at the indicated times. Differences that were not significant are denoted with by ns, P-value <.001 (***); unpaired two-tailed t-test. E, Western blot analysis of PD-L1 and A20 expression in tumor tissues removed from the mice mentioned in A-D. The samples were obtained from two independent experiments. The two groups were exposed to chemiluminescence for the same length of time. F, PD-L1 and A20 expression in the tumor tissues removed from mice was quantified by densitometry. P-value <.01 (**) and <.001 (***); unpaired two-tailed t-test. G, Kaplan-Meier curves from the survival analysis based on Casp8 expression levels in skin melanoma patients and breast cancer patients. The data were downloaded from TCGA and analyzed by R studio software
Supplier Page from Sino Biological, Inc. for Human PD-L1/B7-H1/CD274 Gene ORF cDNA clone expression plasmid, C-His tag