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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Cell Immunol. 2019 Jul 3;344:103949. doi: 10.1016/j.cellimm.2019.103949

Figure 1. PAR4 activation modulates innate immune responses in vitro.

Figure 1.

A) Levels of CXCL10 in cell culture media of bone marrow derived macrophages from WT (white bars) and PAR4−/− (ΔPAR4, black bars) mice in response to the dsRNA mimetic poly I:C (25 μg/mL) and/or thrombin (Thr, 5 nM) were analyzed after 7 hours stimulation. WT BMM (B) or WT murine embryonic fibroblasts (C) were stimulated with poly I:C (25 μg/mL) and/or PAR4 activating peptide (AP, 150 μM) for 7 hours and the CXCL10 protein levels analyzed in cell culture media by ELISA. WT MEF (D, E) were stimulated for 24 hours with poly I:C (25 μg/mL) and/or PAR4 AP (150 μM) and the MCP1 and TNFα protein levels were analyzed in the cell culture media by ELISA. F) TNFα mRNA expression was analyzed by real-time PCR in WT murine CM 4 hours after stimulation with poly I:C (25 μg/mL) and/or PAR4 AP (150 μM). Results are shown relative to poly I:C stimulation which was set to 1. G) CVB3 genomes were analyzed in WT murine CM by RT-PCR after infection with CVB3 (multiplicity of infection 1) and additional stimulation with thrombin (Thr, 5 nM) or PAR4 AP (150 μM) for 6 hours. Genome levels were normalized to HPRT mRNA expression. H) NK cell cytotoxicity against YAC-1 cells of splenocytes isolated from WT (white bars) and ΔPAR4 (black bars) mice after 24 hours stimulation with or without PAR4 AP (150 μM). Data (mean ± SEM; N>4) were analyzed by 2-way (A, H) and 1-way (B-G) ANOVA. Statistical significance is shown as * P<0.05, # P<0.05 versus unstimulated control within the same genotype or unstimulated control, and $ P<0.05 versus poly I:C alone.