Transcriptome analysis indicates early events during necroptosis, suggesting transcriptional dependency. (A) Validation of RIPK1−/− PDX: immunoblot showing efficient knockout of RIPK1, whereas GAPDH is used as a loading control (left), and viability curves indicate response to the SM birinapant in RIPK1+/+ vs RIPK1−/− PDX (right). (B) Schematic representation of experimental setup. (C) Functional enrichment analysis (displayed: Kyoto Encyclopedia of Genes and Genomes [KEGG] pathways) upon 5 hours of SM treatment in PID0117 RIPK1−/− cells (log2FC > 1). (D) Functional enrichment analysis (displayed: KEGG pathways) for the RIPK1-specific signature (log2FC > 0.58). To obtain this signature, log2FC values resulting from SM treatment vs untreated conditions were additionally compared between the 2 isogenic PDXs. Overrepresentation of genes belonging to specific pathways (fold enrichment) is shown on the x-axis; dot sizes represent the number of genes belonging to the specific pathway; FDR is indicated using different colors (green, lower FDR; the enrichment is less likely to be caused by chance). (E) Volcano plot showing differential gene expression (upregulation/downregulation) of genes in RIPK1+/+ cells upon 5 hours of SM treatment. Highlighted are genes upregulated with DNA-binding transcription activity (GO:MF, red) and downregulated with DNA modification activity (GO:MF, blue) in RIPK1+/+ cells upon SM treatment. Horizontal dotted line, −log10 (.05); vertical dotted lines, log2FC (±1). (F) Representation of differentially regulated genes in RIPK1+/+ cells (x-axis) in relation to RIPK1 signature (y-axis) after 5 hours of SM treatment. Colored, labeled dots: genes differentially regulated by |log2FC| >0.58 and P value (adjusted) < .05. (G) TRRUST analysis of differentially regulated genes for RIPK1+/+ cells (log2FC > 1), highlighting the regulators of genes of interest upon 5 hours SM treatment. (H) TRRUST analysis of differentially regulated genes for RIPK1 signature (log2FC > 0.58). RNA sequencing was conducted in triplicates. CTRL, control; FC, fold cange; FDR, false discovery rate; UNTR, untreated.
Figure 3.

Transcriptome analysis indicates early events during necroptosis, suggesting transcriptional dependency. (A) Validation of RIPK1−/− PDX: immunoblot showing efficient knockout of RIPK1, whereas GAPDH is used as a loading control (left), and viability curves indicate response to the SM birinapant in RIPK1+/+ vs RIPK1−/− PDX (right). (B) Schematic representation of experimental setup. (C) Functional enrichment analysis (displayed: Kyoto Encyclopedia of Genes and Genomes [KEGG] pathways) upon 5 hours of SM treatment in PID0117 RIPK1−/− cells (log2FC > 1). (D) Functional enrichment analysis (displayed: KEGG pathways) for the RIPK1-specific signature (log2FC > 0.58). To obtain this signature, log2FC values resulting from SM treatment vs untreated conditions were additionally compared between the 2 isogenic PDXs. Overrepresentation of genes belonging to specific pathways (fold enrichment) is shown on the x-axis; dot sizes represent the number of genes belonging to the specific pathway; FDR is indicated using different colors (green, lower FDR; the enrichment is less likely to be caused by chance). (E) Volcano plot showing differential gene expression (upregulation/downregulation) of genes in RIPK1+/+ cells upon 5 hours of SM treatment. Highlighted are genes upregulated with DNA-binding transcription activity (GO:MF, red) and downregulated with DNA modification activity (GO:MF, blue) in RIPK1+/+ cells upon SM treatment. Horizontal dotted line, −log10 (.05); vertical dotted lines, log2FC (±1). (F) Representation of differentially regulated genes in RIPK1+/+ cells (x-axis) in relation to RIPK1 signature (y-axis) after 5 hours of SM treatment. Colored, labeled dots: genes differentially regulated by |log2FC| >0.58 and P value (adjusted) < .05. (G) TRRUST analysis of differentially regulated genes for RIPK1+/+ cells (log2FC > 1), highlighting the regulators of genes of interest upon 5 hours SM treatment. (H) TRRUST analysis of differentially regulated genes for RIPK1 signature (log2FC > 0.58). RNA sequencing was conducted in triplicates. CTRL, control; FC, fold cange; FDR, false discovery rate; UNTR, untreated.

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