Fig 2: Pbrm1 knockout was associated with less immunogenic TME in murine renal tumor and pre-malignant kidney tissue.a The confidence interval plots represented the differences in these genes between the two groups (Pbrm1 KO minus Pbrm1 WT). The x-axis is the difference in group means of Pbrm1 knockout group minus Pbrm1 wild-type group, so negative values correspond to genes downregulated in the Pbrm1 knockout group. The lines depicted 95% confidence intervals, and genes with a significant difference between the groups (adjusted P-value <0.05) were marked in orange. The P-values were obtained from a two-sample t-test on the log2-transformed values, and the resulting P-values were adjusted for multiple comparisons using the Benjamini–Hochberg method across the gene set. b mRNA expressions of Ifng, Cxcl9, Cxcl10, and Pdcd1 and c the mRNA expression of Pbrm1, Ifngr1, Ifngr2, and Cd274 were detected by real-time PCR. Each dot represents the mean value of triplicated tumor samples. Unpaired t-test was performed with GraphPad Prism 7.03. d The coordinated differences of the genes as listed in (a) across the two groups assessed by gene set enrichment analysis (GSEA). e T cell infiltration and quantification. Murine Renca tumor microarrays, with triplicate formalin-fixed tissue cores for each case, were immunohistochemically stained with antibodies against CD3, CD8, CD4, PD-1, and P-STAT1 Y701. The percentages of positively stained cells were analyzed using inForm software. Unpaired t-test was performed with GraphPad Prism 7.03. Scale bar, 100 µm. f Multiplex Opal Immunofluorescence staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, 520 nm; PD-1, 570 nm). PD-1 Opal signals are artificially colored as red. Scale bar, 50 µm. g Immunomodulatory gene expression signature score in pre-malignant murine kidneys following loss of Vhl alone (Vhl-/-), or Vhl in combination with Pbrm1 (Vhl-/-Pbrm1-/-). Student t-test. h Gene expression-based inference of total T cell and CD8 T-cell infiltrates in pre-malignant murine kidneys following loss of Vhl alone, or Vhl in combination with Pbrm1. Student t-test. Data in the graphs represent mean ± S.D. Source data are provided as a Source Data file.