Fig. 3
KLF14 suppresses IRP2 expression and its suppression is dependent on zinc finger 3. A Human IRP2 promoter luciferase reporter was transfected with KLF14-WT-3 × Flag or KLF14-ΔZF3–3 × Flag (deletion of zinc finger 3) in HepG2 cells for 48 h. Relative luciferase activity was then analyzed. B ChIP assay revealed significant enrichment of KLF14 protein on the IRP2 promoter in HepG2 cells. Schematic diagram of primer pairs of the human IRP2 promoter region (up panel) in the ChIP assay and real-time PCR analysis. HepG2 cells transfected with KLF14-WT-3 × Flag or KLF14-ΔZF3–3 × Flag were harvested (with 3xFlag-Vector as control). Cross-linked samples were immunoprecipitated with anti-FLAG antibody, and the precipitated DNA fragments were subjected to qRT-PCR in the IRP2 promoter regions. C, D Relative mRNA levels of IRP1, IRP2, TfR1 and FH (normalized with GAPDH mRNA) in KLF14-overexpressed (C) or KLF14-silenced (D) HepG2 and Huh7 cells were examined by qRT-PCR. E Western blotting was conducted to test the expression of IRP1, IRP2, TfR1, FH and GAPDH (control) in KLF14-WT-3 × Flag, KLF14-ΔZF3–3 × Flag overexpressed and KLF14-silenced HepG2 and Huh7 cells. F The relative cellular LIP contents in KLF14-WT-3 × Flag and KLF14-ΔZF3–3 × Flag overexpressed cells were measured at 24 h (up panel) and 36 h (down panel). G The cell apoptosis of KLF14-WT-3 × Flag and KLF14-ΔZF3–3 × Flag overexpressed cells were investigated by flow cytometry. H, I Cell growth curve (H) and Colony formation ability (I) of KLF14-WT-3 × Flag and KLF14-ΔZF3–3 × Flag overexpressed cells. All results are presented as means ± SD from three independent experiments. Two-tailed unpaired Student’s T-tests were performed. n.s, not significant, *P < 0.05, **P < 0.01 and ***P < 0.001