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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Gastroenterology. 2015 Nov 12;150(3):707–719. doi: 10.1053/j.gastro.2015.11.002

Figure 4. NANOG and STAT3 influence Twist1 promoter activation in NS5A TICs.

Figure 4

(A) LPS-induces Twist1 promoter activity in TICs. Twist1 promoter analysis with various deletion constructs demonstrated the importance of the TSS proximal segment (nt −209/−1). Relative light units (RLU) values were normalized by Renilla luciferase activity driven by a constitutively active SV40 promoter (pTwist1 nts −1 to −700; *, P<0.05; color matched; pTwist1 nts −1 to −209; #, P<0.05; n=3). (B) NANOG and STAT3 activate the Twist1 promoter. NANOG and STAT3 binding elements in Twist1 promoter region (nts −209 to −51) were mutated by in vitro mutagenesis (pTwist1 1–209WT; *, P<0.05; color matched; n=3). (C) Silencing Tlr4 and Nanog using lentivirus expressing shRNA or Stat3 and Stat3D (retrovirus expressing dominant negative Stat3). *, P<0.05; color matched; n=3). (D) Upper panel, schematic representation (SR) of Twist1 promoter region depicting the locations probed for the consensus binding sequences for NANOG (yellow script), STAT3 (Green lettering), and the specificity control (SC) regions analyzed by ChIP (white script). Immediately below the SR are NANOG ChIP-qPCR (black bar graphs) and STAT3 ChIP-qPCR (blue bar graphs) analyses which demonstrated the enrichment of NANOG and STAT3 in TICs post LPS (10 μg/ml) and leptin (5 ng/ml) treatment. The Fold enrichment values are relative expression values normalized to the IgG controls (SC3; *, P<0.05; SC2; $, P<0.05; SC1; #, P<0.05; biological replicates 4; n=2). (E) Protein-Protein-DNA interaction demonstrated by sequential-ChIP-qPCR, indicated that NANOG and STAT3 bind each other on the Twist1 promoter region in TICs post LPS (10 μg/ml) and leptin (5μg/ml) treatment. The Fold enrichment values are relative expression values normalized to the IgG controls (SC3, SC2, SC1; *, P<0.05; color matched; #, P<0.05; biological replicates 4; n=2).