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. Author manuscript; available in PMC: 2017 Jan 12.
Published in final edited form as: Cell Metab. 2015 Dec 24;23(1):206–219. doi: 10.1016/j.cmet.2015.12.004

Fig. 3. TLR4 signaling transactivates Nanog promoter through E2F1-binding sites.

Fig. 3

(A) Nanog promoter ChIP assay using anti-E2F1 antibody following qPCR in TICs. E2F1 showed enrichment in Nanog promoter.

(B) Truncated promoter luciferase constructs were used to map the region responsive to TLR4 signaling. LPS-mediated Nanog promoter activity is compare with PBS (Vehicle)-treated cells to demonstrate Nanog promoter activity upon TLR4 stimulation (n=3). *: P < 0.05, **: P < 0.01.

(C and D) E2F1-binding sites were required for efficient Nanog transactivation (n=4). Four mutant-luciferase plasmids were constructed by in vitro mutagenesis “M” indicates the sites of mutation.

(D) E2F1 (blue oval) regulated Nanog enhancer for LPS-induced activation.

(E) Overexpression of E2F1 resulted in NANOG promoter activation. Gfp, E2F1 or c-MYC were overexpressed in Huh7 cells and examined for luciferase reporter activities in response to LPS stimulation.

(F) Silencing E2F1 reduced Nanog mRNA and protein levels in response to LPS.

(G) Silencing E2F1 reduced tumor growth in NOG mice.