Fig. 6
KLF14 suppresses the expression of IRP2 by recruiting SIRT1. A Relative mRNA level of IRP2 in KLF14 overexpressed cells and/or NAM treatment. B SIRT1–3 × HA or SIRT5–3 × HA was transfected into 293 T cells with KLF14-WT-3 × Flag for 48 h. Whole-cell lysates were immunoprecipitated with Flag beads and the co-precipitated HA was then detected. C Spatial analysis of KLF14 and SIRT1 or SIRT5 colocalization. SIRT1-RFP or SIRT5-RFP was transfected into 293 T cells with KLF14-EGFP for 48 h. Representative confocal images of KLF14 (green) and SIRT1/SIRT5 (red) in 293 T cells are shown. Scale bar, 10 μm. D Silencing of SIRT1 but not SIRT5 reduced the suppression of KLF14 on IRP2 mRNA level. E The protein level of IRP2 in SIRT1 overexpressed HepG2 and Huh7 cells. F The protein level of IRP2 in KLF14–3 × Flag overexpressed HepG2 cells with SIRT1 silenced. G Stimulated model of three zinc finger domain of IRP2 (green). H Stimulated model of the interaction between the zinc finger domain of IRP2 (yellow) and SIRT1 (blue), and the predicted binding sites of KLF14 (red). I KLF14–3 × Flag (WT), KL14-M1–3 × Flag (mut1: T203A), KL14-M2–3 × Flag (mut2: T203A + S239A), KL14-M3–3 × Flag (mut3: T203A + S239A + R247A) and KL14-M4–3 × Flag (mut4: T203A + S239A + R247A + R254A) transfected with SIRT1–3 × HA into 293 T cells for 48 h, whole-cell lysates were immunoprecipitated with Flag beads and the co-precipitated HA was detected. J KL14-M4–3 × Flag (mut4: T203A + S239A + R247A + R254A) was transfected into HepG2 cells, whole-cell lysates were immunoprecipitated with Flag beads and the co-precipitated SIRT1 was detected. K ChIP assay revealed significant enrichment of KLF14-M4 protein on the IRP2 promoter in HepG2 cells. L Mutation of KLF14 reversed the inhibition effect of KLF14-WT on IRP2 promoter activity. All the 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