Fig. 4: A PYK2 inhibitor Defactinib suppresses IRF5 activation.

a TNF-luc reporter activity in the absence or presence of ectopically expressed IRF5 in wild type, IRF5 KO and PYK2 KO RAW264.7 cells pretreated for 1 h with 1 μM of defactinib (or DMSO control) and then stimulated with 1 ug/ml of LPS for 6 h or left untreated. AU arbitrary units of luminescence. Data were shown as means ± SEM from n = 3 independent experiments. Comparison by two-way ANOVA with Tukey’s correction (*P < 0.05, **P < 0.01 and ***P < 0.001). b RAW264.7 cells were fractionated into cytosolic and nuclear extracted following 1 h pretreatment with defactinib (1 μM) and 2 h stimulation with LPS (1 μg/ml). Representative blots from three independent experiments are shown. c IRF5 and pol II binding to Il6 and Il1b gene promoters in GM-CSF-differentiated mouse BMDMs pretreated with 3.5 μM defactinib (def) or DMSO control and further stimulated with LPS for 2 h. Chromatin recruitment was analysed by ChIP. Data were normalised against chromatin amount in lysates (and expressed as a percentage of input for each gene) and shown as mean values ± SEM from n = 3 independent experiments. Comparison by one-way ANOVA with Tukey’s correction (*P < 0.05, **P < 0.01). d Il6 and Il1b expression levels in GM-BMDMs pretreated with 3.5 μM defactinib (def) or DMSO control for 1 h, followed by LPS stimulation for 2 h. Data were shown as means ± SEM for n = 4 individual mice. Statistical significance was analysed by one-way ANOVA with Tukey’s correction (*P < 0.05 and ***P < 0.001). Source data are provided as a Source Data file.