Figure 4. Upon chronic NOD2 stimulation, expression and cytokine promoter binding of the anti-inflammatory transcription factors c-Maf and Bmi1 are upregulated in a Twist-dependent manner.

(A) MDMs (n=8) were left untreated or pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for 4h (acute). Fold increase in c-Maf, Bmi1 and Akt1 mRNA was normalized to untreated MDMs + SEM. (B) MDMs (n=8) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA, alone or in combination, 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for 4h (acute) and assessed for c-Maf, Bmi1 and Akt1 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (C) MDMs were left untreated or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, or a combination of Twist1 and Twist2 siRNA ± vectors expressing c-Maf or Bmi1, and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 8h (acute) and c-Maf or Bmi1 expression was assessed by Western blot. GAPDH was assessed as a loading control. (D) MDMs were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). Recruitment of c-Maf (n=4) and Bmi1 (n=5) to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (B-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<1×10⁻⁴; ††, p<1×10⁻⁵. Tx, treatment.