Fig. 4: The inhibitory effects of the JNK inhibitor SP in response to IL-1β upregulation, as measured by ELISA assay and western blot analysis.

A ELISA confirmed that CCL5 secretion by P3 ESC from each of four subjects was upregulated after 72 h of IL-1β treatment. This effect was reversed by co-incubation with SP (p < 0.001, ANOVA). Error bars indicate SD of triplicate determinations for subject S4. B When IL-1β was co-incubated with a 100-fold excess of IL-1ra [43], complete abrogation of the IL-1β effect on IL-8 and IL-6 upregulation was observed. The overall comparison using two-factor ANOVA analysis revealed significant differences (p < 0.001). Scheffé Test comparisons showed significant differences between Control vs IL-1β (p < 0.001) and IL-1β vs IL-1ra (p < 0.001). Error bars indicate SD of triplicate determinations. C Time-course of CCL5 ELISA response to IL-1β ± SP (24–72 h). The overall comparison using one-factor ANOVA revealed significant differences between groups (p < 0.001). Error bars indicate SD of triplicate determinations. D Time-course of TNF-α ELISA response to IL-1β ± SP (24–72 h). The overall comparison using one-factor ANOVA revealed significant differences between groups (p < 0.001). Error bars indicate SD of triplicate determinations. E Western blot showing that preincubation of P3 ESC with SP, for 1 h before addition of IL-1β (PSP), had the same inhibitory effect as simultaneous co-incubation of IL-1β plus SP (SSP) on IL-1β, IL-6, TNF-α and MMP3 reduction. SP alone reduced basal (control) SASP markers. β-Actin levels were not affected. F Western blot reveals inhibition of IL-1β stimulation of IL-1β, IL-6, MMP3, p21 and HMGB1 by JNK1 (CJ1) or JNK2 (CJ2) siRNA constructs transfected into P3 ESC. β-Actin levels were not affected.