|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 6. Hint1 represses TCF-4–ß-catenin transcriptional activity. Reporter gene assays were performed in HEK293 cells transiently transfected with different combinations of plasmids as indicated. Data represent the mean of at least four independent transfections each measured in duplicate. (A) The improved Topflash/Fopflash reporter constructs pGL3-OT/OF were used to measure the effects of Hint1 on TCF-4–ß-catenin transcriptional activity. Cells were transfected with 1.0 µg pGL3-OT/OF, 0.7 µg pCS2+Reptin52 or pCS2+Pontin52, 0.5 µg pcDNAI-hTCF4, 0.5 µg pCS2+ß-catenin, 1 µg pCS2+Hint1 and 0.1 µg pCH110. (B) The repressive effect of Hint-1 on TCF-4–ß-catenin mediated transcription was also detectable with the Siamois luciferase reporter construct (1.0 µg). Increasing amounts of Hint1 (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg) expression plasmid were transfected with constant amounts of TCF-4, ß-catenin and Pontin or Reptin plasmids. Transfection of an equal amount of empty pCS2+ vector was used as a control. (C) LEF-VP16 (1.0 µg) driven transcription is not repressed by Hint1. 
N-LEF-1 (1.0 µg) without ß-catenin binding site does not activate transcription of the reporter gene. (D) Reporter gene activity was measured in NIH3T3 cells stably expressing Wnt-1 and control cells after transient transfection with Hint1 (2 µg). (E) Wnt-1 expressing C57MG cells show reduced reporter gene activity when transfected with Hint1 (2 µg). (F) SW480 colon carcinoma cells were transiently transfected with increasing amounts (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg) of Hint1 and TCF-4–ß-catenin transcriptional activity was analysed with the Siamois luciferase reporter system. In A-C pCH110 (ß-galactosidase) and in D-F pHRL-TK (Renilla luciferase) was used for normalization. S5, wild-type Siamois promoter; S0, Siamois promoter with mutated LEF-1/TCF binding sites.
|
