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. 2015 Oct 6;6:8450. doi: 10.1038/ncomms9450

Figure 4. KLF14 inhibits Plk4 expression.

Figure 4

(a–d) KLF14 overexpression inhibits Plk4 transcription and protein expression. (a,b) Human Plk4 promoter (−1343 to −141 bp upstream of ATG) luciferase reporter was co-transfected with indicated Flag-tagged KLFs (a) or KLF14-ΔZF2 (deletion of zinc finger 2) (b) in HeLa cells for 24 h, then relative luciferase activity was analysed. Data represent mean±s.d., **P<0.01. Representative western blotting showing the expression of the Flag-tagged KLFs detected with anti-Flag antibody. (c,d) HeLa and U2OS cells were transfected with Flag-KLF14 (WT) or Flag-KLF14-ΔZF2 (ΔZF2) for 36 h. The mRNA levels of Plks (Plk1-4) and β-actin (as internal standard) were analysed by reverse transcription–PCR (RT–PCR) (c). Protein levels of Flag-KLF14 and Plk4 were examined by western blotting using anti-Flag and anti-Plk4 antibodies, respectively (d). Relative levels of Plk4 mRNA and protein bands were quantified using densitometric analysis. (e,f) KLF14 knockdown increases Plk4 expression. HeLa, U2OS and HCT116 cells were infected with pLKO.1 vector (Control) or KLF14-knockdown (Si-1 or Si-2) lentivirus for 72 h. The mRNA and protein levels of endogenous Plk4 and KLF14 were examined by RT–PCR (e) and western blotting (f). Relative levels of Plk4 mRNA and protein bands were quantified using densitometric analysis. (g–i) KLF14 KO increases Plk4 expression. (g,h) The protein and mRNA levels of Plk4 in WT and KLF14-KO passage 3 (P3) MEFs were examined by western blotting (g) and RT–PCR (h). Relative levels of Plk4 mRNA and protein bands were quantified using densitometric analysis. (i) Plk4 protein levels in indicated tissues of KLF14-WT and -KO littermate mice were examined by western blotting. (j) KLF14 binds to Plk4 promoter. HeLa cells transfected with vector (Control), Flag-KLF14 (WT) or Flag-KLF14-ΔZF2 (ΔZF2) were processed for chromatin immunoprecipitation using FLAG M2 beads. Co-precipitated chromatin DNA was analysed by PCR using a pair of primers that amplify the −463 to −318 bp region of Plk4 promoter.