FIG 5.

Identification of the BHLHE40/41 responsible site in the TWIST1 promoter. (A) Reporter analysis of the SNAI1, SNAI2, and TWIST1 promoters in Ishikawa, HEC-1, and HEC-6 cells transfected with vectors to express HA-BHLHE40 and/or FLAG-BHLHE41. (B) Reporter analysis of the SNAI1, SNAI2, and TWIST1 promoters in HHUA cells transfected with the vectors to knock down BHLHE40 and/or BHLHE41. (C) Truncation assay of TWIST1 reporters. Five kinds of reporters possessing upstream regions from bp −1876, −1587, −170, +34, and +116 from the transcription start site were analyzed for their activity. (D) The pTWIST1+116 reporters possessing a deletion or mutations at the SP1 binding site (SBS) were used to analyze their activity. (E) The control activity of the mutant pTWIST1+116 reporter was adjusted to the same value as that of the wild-type reporter to evaluate the effects of BHLHE40/41 expression (white bars). (F) Impact of the SBS in the full-length TWIST1 reporter was evaluated using the pTWIST1-1876 reporter possessing a mutation at the SBS. The control activity of the mutant pTWIST1-1876 reporter was adjusted to the same value as that of the wild-type reporter to evaluate the effects of BHLHE40/41 expression (white bars). n.s., not significant; *, P < 0.05; **, P < 0.01. #, P < 0.05; ##, P < 0.01 (significantly different from the pCDNA3 control). §, P < 0.05; §§, P < 0.01 (significantly different from the pCDNA3 control).