Fig. 5

Enhancement of granzyme B toxicity with IL-1β. a IL-1β (20 ng/ml) augments granzyme B neurotoxicity. Cumulative neurotoxicity of combined granzyme B and IL-1β reaches 100%. ****p < 0.0001 cumulative toxicity relative to control media, ####p < 0.0001 cumulative toxicity relative to granzyme B + IL-1β (two-way ANOVA with post hoc Tukey’s test, n = 5/condition. Data are mean ± standard error of the mean from three separate experiments). b IL-1β is not toxic by itself. The IL-1 receptor antagonist AF 12198 (1 μM) abolished IL-1β effect after 3 days of co-culture with granzyme B and IL-1β (20 ng/ml). ****p < 0.0001 relative to control media, ####p < 0.0001 between granzyme B + IL-1β and granzyme B+ IL-1β + IL-1 receptor antagonist, repeated measures ANOVA with post hoc Tukey’s test, n = 6/condition. Data are mean ± standard error of the mean from three separate experiments. c After 3 days of exposure, IL-1β (20 ng/ml) significantly increased PAR1 mRNA as shown by qPCR and PAR1 protein relative to control levels using Western blot analysis. The IL-1 receptor antagonist AF 12198 (1 μM) blocked the IL-1β effect on PAR1 mRNA and protein. A pooled PAR1 siRNA preparation reduced PAR1 mRNA and protein below control conditions. d Western blot analysis (below bar graph) confirmed the relative amounts of PAR1 protein in respective conditions. *p < 0.05, ****p < 0.0001 relative to control media, one-way ANOVA with post hoc Tukey’s test, n = 4/condition. Data are mean ± standard error of the mean from duplicate experiments. e Granzyme B had no toxicity when PAR1 siRNA reduced PAR1 protein levels. A pooled PAR1 siRNA preparation inhibited all granzyme B neurotoxicity; IL-1β had no effect. Two control siRNA, anti-GAPDH, and a scrambled PAR1 sequence had no effect. ****p < 0.0001 relative to control media, ####p < 0.0001 between PAR1 siRNA + granzyme B+ IL-1β and other siRNA as noted, one-way ANOVA with post hoc Tukey’s test, n = 8/condition. Data are mean ± standard error of the mean from four separate experiments