Fig. 4

The RhoA/Rock Inhibitor Y27632 downregulates type I IFN signaling. A HEK293T cells were co-transfected with ISRE-luciferase and Renilla reporter plasmids for 24 h, then cultured with or without Y27632 (60 μM, 45 min) before the addition of IFN-a (1000 U/mL, 6 h). Luciferase activity was measured by dual luciferase assay. B PBMCs from healthy controls were cultured in the presence or absence of Y27632 (60 μM, 45 min) and stimulated with IFN-a (1000 U/mL) for different times. Western blots show whole-cell lysates harvested at the indicated times. C Histograms show the ratios of phosphorylated to total STAT-1 at the indicated times. The ratio of pSTAT-1/STAT-1 in the absence of Y27632 and IFN-a at 0 min was set at 1. D Relative expression of OAS1 and E CXCL10 mRNA in PBMCs cultured with Y27632 (0, 30, 60, and 90 μM, for 45 min and stimulated with IFN-a (1000 U/mL) for 6 h. Results are the relative expression levels of OAS1 and CXCL10 mRNA normalized to endogenous GAPDH mRNA levels. F CXCL10 levels in PBMC culture supernatants quantified by ELISA. Bars show the mean ± SEM of 3 individual healthy donors; *p < 0.05, **p < 0.01, A and C versus vehicle by Student’s t-test