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. 2017 Nov 27;8(65):109301–109318. doi: 10.18632/oncotarget.22673

Figure 7. Transcriptional regulation of the Ascl2 gene in YAP1-interfered CD133+ CD44+ CRC cell population or YAP1-enforced expressed HT-29 or Caco-2 cells.

Figure 7

We performed luciferase assays using YAP1-interfered CD133+CD44+ CRC cell population to analyze the transcriptional regulation of the Ascl2 gene. (A) We produced a series of pGL3-Basic firefly luciferase reporters containing different human Ascl2 promoters encompassing bp -2588 to +620 (pGL3-p3), -628 to +620 (pGL3-p2), and -82 to +620 (pGL3-p1). The black dots represent GC-boxes. (B and C) A reduction of Ascl2 promoter activity was evident from the luciferase reporter assays in YAP1-interfered CD133+CD44+ HT-29 (B) and Caco-2 (C) cell population compared with their controls (n=3) (**: P<0.01). (D) A schematic representation of human Ascl2 promoter (-628 to +620) constructs containing the second potential KLF5 binding site, GC-box (GGGCGG) (GC-box wt), that was used in this study; the GC-box was mutated to GC-box mutant (AAGTAG) (GC-box mt). (E and F) YAP1-interfered CD133+CD44+ HT-29 (E) and Caco-2 (F) cell population and their control cells were transfected with the Ascl2 promoter-Luc construct (GC-box wt) or its mutant (GC-box mt). Reduced Ascl2 promoter activity was evident based on luciferase reporter assays (n=3) (**: P<0.01). (G and H) A significant increase in Ascl2 promoter activity was evident based on the luciferase reporter assays in lv-YAP1/HT-29 (G) and lv-YAP1/Caco-2 (H) cells compared with their controls (n=3), KLF5 interference in lv-YAP1/HT-29 (G) and lv-YAP1/Caco-2 (H) cells led to the significant reduction of Ascl2 promoter activity compared with their control cells (**: P<0.01). (I and J) The lv-YAP1/HT-29 (I) and lv-YAP1/Caco-2 (J) cells and their control cells were transfected with the Ascl2 promoter-Luc construct (GC-box wt) and its mutant (GC-box mt). GC-box mutation led to the significant reduction of Ascl2 promoter activities based on the luciferase reporter assays (n=3), KLF5 interference in lv-YAP1/HT-29 (I) and lv-YAP1/Caco-2 (J) cells led to further reduction of Ascl2 promoter activity compared with their control cells which were transfected with the Ascl2 promoter-Luc construct (GC-box wt) or its mutant (GC-box mt) (**: P<0.01).