FIGURE 1.

Transcriptomic profiling reveals a novel gene set specifically regulated by RIPK2 kinase activity. A, primary BMDMs were stimulated with vehicle or with each of the RIPK2 kinase inhibitors Gefitinib or SB203580, in the absence or presence of, the NOD2 agonist MDP. RNA was extracted from each condition and subjected to RNA sequencing. While Gefitinib inhibits both RIPK2 and the EGF-R, and SB203580 inhibits both RIPK2 and p38, the only shared “off-target” kinase they inhibit to the same extent as their intended target is RIPK2. Focusing on transcripts differentially expressed by both RIPK2 inhibitors during NOD2 activation allowed us to determine 26 genes that were specifically regulated by RIPK2 kinase activity. B, of the genes identified, pathway analysis showed that the RIPK2 kinase-dependent genes were preferentially involved in immune functions, cell metabolism and nucleotide/nucleoside regulation. C, of the RIPK2 kinase-dependent genes identified using RNA sequencing, 9 genes were selected and validated using qRT-PCR. Using BMDMs, all 9 genes showed significant up-regulation in the presence of MDP and a corresponding down-regulation when either of the 2 RIPK2 inhibitors, Gefitinib or SB203580, were present. Experiments were performed in duplicate on three separate occasions (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).