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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: J Immunol. 2015 May 27;195(1):217–226. doi: 10.4049/jimmunol.1402808

Figure 6. Expression and cytokine promoter binding of the transcriptional activators ATF4, C/EBPα, Runx1 and Runx2 is reduced in a Twist-dependent manner during chronic NOD2 stimulation.

Figure 6

(A) MDMs (n=8) were left untreated or pre-treated with 100μg/ml MDP for 48h, and then MDMs were treated with 100μg/ml MDP for 4h (acute). Fold change in ATF4, C/EBPα, Runx1, Runx2 and Akt2 mRNA 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, and 24h later (total 48h MDP treatment), MDMs were stimulated with 100μg/ml MDP for 4h (acute) and assessed for ATF4, C/EBPα, Runx1, Runx2 and Akt2 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (C) MDMs (n=4-6) 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 MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). Recruitment of ATF4, C/EBPα, Runx1 and Runx2 to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA-transfected cells + SEM. (B-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p<0.01; **, p<0.01; ***, p<0.001; †, p<1×10−4. Tx, treatment.