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Fig. 4. Klf5 is a component of the Oct3/4 and Nanog transcription-factor network. (A) Nanog-Luc or Pou5f1-Luc were co-transfected with Klf5 (Klf5 ov.), Klf5 shRNA or control (mock) vectors. (B) 3xFLAG-Klf5 transfected ESCs were processed for ChIP with antibody for the FLAG epitope or IgG as control. The amount of precipitated DNA was calculated relative to the total input chromatin. (C) The indicated oligonucleotide, designed on the basis of the Nanog promoter (Wu and Yao, 2005), was incubated with nuclear extracts of mock-transfected (lanes 2-4) or 3xFLAG-Klf5-transfected (lanes 6-9, 11,12) cells. Nuclear extracts were preincubated with 50-fold molar excess of unlabelled oligo probe (lanes 3 and 7) or unrelated oligo (lanes 4 and 8), or mutant oligo in which the sequence –78 to –72 (GGGTGGG, underlined) was changed into AGATAGA to disrupt the candidate cis-element of Klf5. In lane 12, nuclear extracts were preincubated with anti-Klf5 antibody. Relevant bands are indicated (arrows) as follows: (a) candidate band for endogenous Klf5; (b) specific shifted band not related to Klf5; (c) additional band due to exogenous 3xFLAG-Klf5. (D) ChIP experiments with anti-Nanog antibody on the Klf5 promoter. K1 and K2 indicate two oligonucleotide pairs that amplify the regions of the Klf5 gene (supplementary material Fig. S7). The differences with control antibody and control DNA were significant (P<0.001). (E) Real-time PCR of Klf5, Nanog, Oct3/4 and Sox2 mRNAs in Klf4-KD or -overexpressing cells. ^*P<0.001, ^**P<0.01. (F) mRNA levels of Klf2 and Klf4 in ESCs transfected with Oct3/4, Nanog or Klf5 shRNAs or with Klf5 overexpressed (Klf5 ov). ^*P<0.001.

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