Figure 5. Effects of RIPK2 PROTAC on LPS-induced pro-inflammatory gene expression, protein levels of iNOS and COX-2, and phosphorylated NF-κB p65 and MAPK p38 levels in SIM-A9 cells.

SIM-A9 cells were pretreated with 1 μM RIPK2 PROTAC for 4 h followed by 20 h incubation with 10 ng/mL lipopolysaccharide (LPS). After that, cells were harvested for RNA and protein extraction. A) Graph shows that RIPK2 PROTAC partly reduced LPS-induced Ptgs2, Il-1β, Il6, Ccl2 and Mmp9 gene transcription, but did not affect the gene expression of Nos2 and Tnfα. B-E) RIPK2 PROTAC had no effects on the LPS-induced increase in iNOS protein levels (B, C), but it slightly attenuated the LPS-induced upregulation of COX-2 levels (B, D). Treatment with RIPK2 PROTAC had no effects on increased levels of phosphorylated NF-κB p65 (B, E) or p38 MAPK (B, F) induced by LPS. At the same time, LPS-triggered upregulation of RIPK2 was potently degraded by its PROTAC (B, G). qRT-PCR data are normalized to the reference genes Cyc1 and Rltr2aiap and represented as fold changes compared to LPS treatment, and immunoblot data are normalized to β-actin. One-way ANOVA with Bonferroni post-test, *P<0.05, **P<0.01 and ***P<0.001. Data are represented as mean ± SEM from three independent experiments.