Rosiglitazone Ameliorates Spinal Cord Injury via Inhibiting Mitophagy and Inflammation of Neural Stem Cells

Figure 6

Identification of the core sequences on transcription regulation in the PINK1 gene. (a) Dual-luciferase reporter gene assay results showing region of interest in PINK1 gene increased luciferase activity. The whole region (−2300 bp accounting from the translation site and the first intron) was used. (b) The schematic illustrated three reporters divided from the whole region of interest in PINK1 gene (upper). Dual-luciferase reporter gene assay results showing the effects of the respective reporter in luciferase activity (lower). (c) The schematic illustrating FOXO1 binding sites in the first intron of PINK1 gene predicted by GenomatrixMatInspector. Putative core promoter elements around FOXO1 binding sites were proposed by GPMiner. (d) Dual-luciferase reporter gene assay showing promoter deletion analysis of reporter 3, which contains three FOXO1 binding sites. (e) Effects of FOXO1 binding site mutations on luciferase activity. Control: FOXO1 was induced to expression, and DMSO was added instead of Rosi. (f) The schematic illustrating regulatory elements for transcription in PINK1 gene (upper). Yellow, exons; Black, the first intron. The vertical line, FOXO1 binding site 1 (△); transverse line, FOXO1 binding sites 2 and 3 (▽); green with black blocks inside, TATA box (▷). (g) Results of CHIP analysis using a FOXO1 antibody. Data represent three independent experiments and are expressed as (a, b, d–f). versus basic vector (a, b), the 2371 segment (d), the control (e), or the DMSO treatment (f); ^# versus basic vector (d) or Mu-1 (e). UTR: untranslated region; FCS: FOXO1 consensus sequences; Mu_1, Mu_2, Mu_3, Mu_23, and Mu_all denote mutations of FCS1, FCS2, FCS3, FCS2/3, and FCS 1/2/3, respectively.

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