Panel A shows a volcano plot with log2 fold change on the x-axis and −log10 P-value on the y-axis, highlighting genes upregulated in knockout and wild-type conditions. Panel B shows a second volcano plot with the same axes and color-coded gene distributions, highlighting genes upregulated in knockout and wild-type conditions. Panel C shows a dot plot of GeneRatio and normalized enrichment score for selected inflammatory and signaling pathways, with dot size representing count. Panel D shows a bar graph of tumor necrosis factor alpha expression relative to RPL0 for wild type, wild type plus tumor necrosis factor alpha, tumor necrosis factor receptor 1 knockout, and tumor necrosis factor receptor 1 knockout plus tumor necrosis factor alpha. Panel E shows flow cytometry histograms of CD45-negative and CD45-positive populations for isotype control, EO771, MC38, and B16F10 conditions. Panel F shows eight bar graphs measuring CXCL1, CXCL2, CCL2, and CSF-1 concentrations in picograms per milliliter for MC38 and EO771 conditions across the indicated groups. Panel G shows two bar graphs measuring prostaglandin E synthase in nanograms per milliliter for MC38 and EO771 conditions across the indicated groups. Panel H shows two bar graphs measuring prostaglandin E2 in picograms per milliliter for MC38 and EO771 conditions across the indicated groups.
TNFα induces the expression of multiple secondary inflammatory mediators in mouse tumor cells. (A and B) Volcano plots showing genes induced or downregulated by TNFα in EO771 cells, as assessed by bulk RNA-seq 6 and 24 h following TNFα exposure of the cultures. The genes involved in pro-tumor inflammation are highlighted in purple. (C) Enrichment plot showcasing gene sets upregulated, including multiple proinflammatory pathways. (D) TNFα gene expression levels relative to RPL0 mRNA levels assessed by quantitative RT-PCR in WT and TNFR1KO variants. (E) Expression of membrane and intracellular TNFα in cell suspensions from the indicated excised tumors, gated on CD45− (left histograms) and CD45+ cells (right histograms). (F) CXCL1, CXCL2, CCL2, and CSF-1 concentrations in the indicated 72 h cell culture supernatants of MC38 and EO771 cells after induction with rTNFα (n = 6). (G) Induction of PGE synthase protein assessed by ELISA in the same culture supernatants (n = 6). (H) PGE2 concentrations in supernatants measured by ELISA (n = 6). Bulk mRNA-seq was performed with three replicates, and experiments in D–F were analyzed by Mann–Whitney U test and performed at least twice with comparable results. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).