Figure 3
From: PITX1 inhibits the growth and proliferation of melanoma cells through regulation of SOX family genes
Upregulation of SOX9 promoter transcriptional activity by PITX1. (A) The schematic drawing shows the position of the truncation site of each SOX9 reporter plasmid. Black, gray and white boxes indicate PITX1 regulatory elements (RE). (B) PITX1 enhanced relative luciferase activity in a dose-dependent manner. The firefly luciferase activity (Pho) was standardized using Renilla reniformis luciferase activity (Ren) from co-transfected pGL4.70. Luciferase activity in empty-vector-transfected cells (PITX1:0ng) was arbitrarily set at 1. Data are presented as means ± S.D. for three independent experiments. (C) PITX1 expression vectors were co-transfected into the human melanoma cell line A2058 with reporter plasmids containing a full-length promoter (SOX9pro − 1100), or a truncated SOX9 promoter region in which RE sites in the SOX9 promoter region partly or fully eliminated (SOX9pro − 540 and SOX9pro − 486). Luciferase activity in empty-vector-transfected cells was arbitrarily set at 1. Data are presented as means ± S.D. for three independent experiments (*P < 0.05, ***P < 0.001). (D) The schematic diagram shows the luciferase reporter plasmids that encode wild-type and RE mutated versions of the SOX9 promoter region (RE1 mut, RE2 mut and RE3 mut). The cross mark represents mutation. The del3 reporter plasmids are deleted of RE1, RE2 and RE3 in the SOX9 promoter region. (E) PITX1 expression vectors were co-transfected with reporter plasmids containing the wild-type, RE-mutated, or deleted versions of the SOX9 promoter. Luciferase activity in empty-vector-transfected cells was arbitrarily set at 1. Data are presented as means ± S.D. for three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). (F) Schematic diagram showing the SOX9 promoter and three boxes indicating PITX1 binding regions (upper panel). The black arrows indicate qPCR primers for the RE region in the SOX9 promoter based on a ChIP assay. ChIP assay of RE regions in the SOX9 promoter, showing enrichment with PITX1 antibody compared with IgG controls in A2058 clones. Anti-IgG antibody was used as a negative control. Input represents qPCR for the SOX9 promoter DNA before immunoprecipitation. The data represent the ratio of the target fragment to the input DNA. Bars correspond to means ± S.D. for three independent experiments (P = 0.01, ns: not significant). All the bar graphs were created using Excel.
