Figure 9. LACC1 regulates NOD2-induced polyphenol oxidase activity in MDMs.

(a) MDMs were transfected with scrambled or LACC1 siRNA, and then treated with 100 μg ml⁻¹ MDP for 6 h and polyphenol oxidase activity was assessed in cell lysates utilizing syringaldazine, ABTS, genistein and resveratrol as substrates (n=4). Similar results were seen in an additional n=4 for syringaldazine and ABTS. (b–e) MDMs were preincubated with kojic acid, salicylhydroxamic acid (polyphenol oxidase inhibitors) or a Syk inhibitor for 1 h, and then stimulated with 100 μg ml⁻¹ MDP and assessed for: (b) polyphenol oxidase activity at 6 h (n=8, similar results were seen in an additional n=4), (c) mtROS and cellular ROS at 6 h (n=6, similar results were seen in an additional n=4), (d) cytokines at 24 h (n=4, similar results were seen in an additional n=8) and (e) bacterial clearance (n=4; significance is compared to non-MDP-treated MDMs without inhibitor treatment or as indicated). Curdlan (cur) treatment is used as a control in b,d. (f) LACC1 Ile254, Val254 or His249,250Ala variants were transfected into HEK293 cells along with NOD2. Cells were then treated with 100 μg ml⁻¹ MDP for 6 h. Polyphenol oxidase activity was assessed. Data represent three replicates repeated two independent times. Inh, inhibitors; salic, salicylhydroxamic acid; Tx, treatment. *P<0.05; **P<0.01; ***P<0.001; ^†P<1 × 10⁻⁴; ^††P<1 × 10⁻⁵; NS, not significant; determined by 2-tailed t-test.