Fig 1: Synergistic effect of Prdm1 overexpression and PD-1 mAb therapy in mice.a, b Schematic view of the treatment plan in subcutaneous tumors and orthotopic tumors. C57BL/6 mice were implanted with Hepa1-6/H22 cells subcutaneously or as orthotopic tumors and were treated with PD-1 mAb or IgG isotype control. c, d Representative xenograft tumors (left) and orthotopic tumors (right) obtained after euthanizing the mice. e, f Tumor proliferation curves (e) and Kaplan–Meier survival curves (f) of Hepa1-6-bearing C57BL/6 mice. Data presented as mean ± SEM (e). n = 6 mice per group. g, h Tumor proliferation curves (g) and Kaplan-Meier survival curves (h) of H22-bearing C57BL/6 mice. Data presented as mean ± SEM (g). n = 6 mice per group. i, j Flow cytometry analysis of CD8+, GZMB+CD8+, and PD1+CD8+ in CD3+ TILs from Hepa1-6 (i) or H22 (j) subcutaneous tumors in C57BL/6 mice. Data presented as mean ± SEM. n = 6 mice per group. k A schematic view of the treatment plan in the DEN/CCL4-induced HCC model. l Representative images of liver tumors (annotated by white arrows). m H&E staining of liver sections of sacrificed mice. Scale bars, 400 µm. n, o Quantification of tumor numbers (n) and maximal tumor sizes (o). Data presented as mean ± SEM. n = 6 mice per group. p Kaplan–Meier survival curves in the DEN-induced HCC model. n = 6 mice per group. P value was determined by unpaired two-sided Student’s t test (e, g, i, j, n, o) and Kaplan–Meier method (f, h, p) with no correction for multiple comparisons. Schematic diagrams (a, b, k) were created with BioRender.com. Source data are provided as a Source data file.
Fig 2: USP22 inhibits ubiquitin-proteasomal degradation of SPI1.a, b SPI1 protein expression in PRDM1-overexpressing and vector Hep3B cells (a) or in PRDM1-knockout and sgCtrl Huh7 cells (b). n = 3 independent biological replicates. c SPI1 protein expression was determined in PRDM1 knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. d SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. e SPI1 was pulled down and ubiquitin-conjugated SPI1 was determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. f DUB siRNA library screening indicated that USP22 and USP33 suppression decreased SPI1 protein levels. g qRT-PCR of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Western blotting of USP22 or USP33 expression in PRDM1-overexpressing and vector Hep3B cells or in PRDM1-knockout and sgCtrl Huh7 cells. n = 3 independent biological replicates. i, j SPI1 protein expression in USP22-overexpressing and vector Hep3B cells (i) or in USP22-knockdown and control Huh7 cells (j). n = 3 independent biological replicates. k SPI1 protein expression was determined in USP22-knockdown or control Huh7 cells. n = 3 independent biological replicates. l SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. m SPI1 was pulled down and ubiquitin-conjugated SPI1 was then determined by immunoblotting using an anti-HA antibody. n = 3 independent biological replicates. P value was determined by unpaired two-sided Student’s t test (g). Source data are provided as a Source data file.
Fig 3: USP22 was identified as a SPI1-interacting protein.a Co-IP experiments indicated the interaction of endogenous USP22 and SPI1. n = 3 independent biological replicates. b Confocal microscopy showing colocalization of USP22 (red) with SPI1 (green). n = 3 independent biological replicates. Scale bars, 20 µm. c Schematic illustration of USP22 and its mutants. d USP22 and its mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. e SPI1 ubiquitination was determined by SPI1 immunoprecipitation and western blotting using an anti-HA antibody. n = 3 independent biological replicates. f Schematic illustration of USP22 and its point mutants. g USP22 and its point mutants were immunoprecipitated and the bound SPI1 was determined. n = 3 independent biological replicates. h The effects of USP22 and its point mutants on SPI1 ubiquitination were confirmed. n = 3 independent biological replicates. i The protein levels of USP22 (Myc) and SPI1 were detected. Data presented as mean ± SEM (n = 3 independent biological replicates). j Schematic representation of full-length SPI1 and truncated SPI1. k Interactions between USP22 and full-length or truncated SPI1 were analyzed using co-IP in HEK-293T cells. n = 3 independent biological replicates. l Co-IP experiments indicated the interaction of Myc-USP22 (161-525) and His-SPI1 (D3) in HEK-293T cells. n = 3 independent biological replicates. m Putative BLIMP1-binding site (BBS) within the genomic sequence adjacent to TSS of USP22 gene. n, o Luciferase activities of USP22 promoter reporter vectors in Hep3B (n) and Huh7 (o) cells. Red characters in the binding regions suggest putative or mutated BLIMP1 binding sequences. Data presented as mean ± SEM (n = 3 independent biological replicates). p, q ChIP analysis of BLIMP1 binding to the USP22 promoter in Hep3B (p) and Huh7 (q) cells. Two promoter regions of USP22 not expected to be bound by BLIMP1 were employed as negative controls. Data presented as mean ± SEM (n = 3 independent biological replicates). P value was determined by unpaired two-sided Student’s t test (n, o, p, q). Source data are provided as a Source data file.
Fig 4: Single-cell analysis of intra-tumoral cell populations.a In total, 8 cell types were identified and shown using UMAP. The colors were coded by cell types (top) and patients (bottom). b Dot plot showing the top marker genes of each cell type. c UMAP plots (top) and PRDM1 expression (bottom) in malignant cells from two patients. d UMAP plot and violin plot showing distinct T cell subclusters and their marker genes. e Dot plot depicting the relative frequency of each T cell subpopulation between patient 1 and patient 2. f The top 10 GO terms of upregulated genes in PRDM1-high tumor cells from patient 2.
Fig 5: PRDM1 upregulates PD-L1 expression in HCC.a qRT-PCR of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Data presented as mean ± SEM (n = 3 independent biological replicates). b, c Western blotting of PD-L1 expression in Hep3B cells or Huh7 cells with or without IFN-γ treatment. Representative of n = 3 independent biological replicates. d, e Flow cytometry analysis of PD-L1 expression in Hep3B cells (d) or Huh7 cells (e) with or without IFN-γ treatment. n = 3 independent biological replicates. f 3D-co-culture model using pre-activated T cells and Hep3B or Huh7 cells. g Flow cytometry analysis of CD8+GZMB+ cell content in pre-activated T cells following 72 h of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). h Flow cytometry analysis of CD8+TNFα+ cell content in pre-activated T cells following 72 hours of co-culture with Hep3B or Huh7 cells. Data presented as mean ± SEM (n = 3 independent biological replicates). i Time-lapse microscopic images revealing the binding of Hep3B or Huh7 cells with PD1 at 12 h. Scale bars, 100 µm. j Analysis of PD1/Fc protein binding on Hep3B and Huh7 cells at 2 h. Data presented as mean ± SEM (n = 3 independent biological replicates). k Immunohistochemical staining of BLIMP1 protein in HCC samples (Cohort 1). Scale bars, 100 µm. l Correlation analysis of tumoral BLIMP1 protein expression and tumor-infiltrating T cell content. Pearson analysis revealed a negative correlation between tumoral BLIMP1 protein expression and CD8+ T cells and the activity (GZMB+) of infiltrated CD8+ T cells in infiltrating CD45+ cells and a positive correlation between tumor BLIMP1 protein expression and the percentages of PD1+ cells in CD8+ TILs. n = 40 patients. P value was determined by unpaired two-sided Student’s t test (a, c, g, h, j) and Pearson correlation analysis (l). Source data are provided as a Source data file.
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