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