Fig. 2: DENR depletion impaired the JAK-STAT signaling pathway.

a Western blotting validation of DENR knockout and PD-L1 expression with two specific sgRNAs in MC38 cells, ± IFNγ (20 ng/ml) for 24 h. The experiment was repeated independently three times with similar results. b Surface PD-L1 levels were analyzed by FACS in DENR and PD-L1 KO cells, + IFNγ (20 ng/ml) for 24 h (n = 3 independent samples). Results are representative of three biological replicates. Three technical replicates are shown. Data are presented as mean values ± SD. Two-sided, one-way ANOVA with Dunnett’s post hoc test: *** p < 0.001. P values from left to right: <0.0001, <0.0001. c RT-qPCR analysis of PD-L1 mRNA levels normalized to β-actin mRNA in control and DENR KO MC38 cells, ± IFNγ (20 ng/ml) for 4 h (n = 3 biologically independent samples). Data are presented as mean values ± SD. Two-sided, one-way ANOVA with Dunnett’s post hoc test: *** p < 0.001, ns: not significant. P values from left to right: 0.1059, > 0.9999, < 0.0001, < 0.0001. d Volcano plot of global gene expression in DENR KO versus control MC38 cells (n = 2 independent samples) upon IFNγ stimulation (20 ng/ml for 4 h). Upregulated genes are labeled as red dots, whereas downregulated genes are labeled in blue. e KEGG analysis of downregulated genes in DENR KO cells versus control cells following IFNγ treatment. f Gene Set Enrichment Analysis (GSEA) showing downregulation of JAK-STAT, and PD-L1 pathways in DENR KO cells. p value is calculated using GSEA Empirical phenotype-based permutation test, two-sided, and no adjustments were made for multiple comparisons. g Western blotting analysis of STAT1/3, p-STAT1/3, CDK4, and NF-kB expression in control or DENR KO MC38 cells, ± IFNγ (20 ng/ml) for 1 h. The experiment was repeated independently three times with similar results. Source data are provided as a Source Data file.