Figure 4

Effects of TRABID shRNAs or overexpression on Wnt reporter gene activity and Axin2 expression. A) Effects of TRABID shRNAs on TRABID expression. HEK293T cells were cotransfected with GFP (an internal control), FLAG-TRABID, and one of TRABID shRNA. Western analyzed was performed 48 hours after transfection. B) TRABID shRNA knockdown efficiency. HCT116 cells were transfected with shRNAs and sorted for YFP positive cells. As a control, HCT116 cells were transfected with the TRABID siRNA (siTrbd). Relative TRABID mRNA levels were determined by q-RT-PCR. C,D) Effects of TRABID shRNAs on Wnt reporter gene activity. HCT116 (B) or SW480 (C) cells were cotransfected with FOPFLASH or TOPFLASH, Renilla luciferase, and one of the TRABID shRNAs. TOPFLASH activity was detected 72 hrs after transfection. E) Effects of TRABID shRNAs on Axin2 expression in SW480 cells. Cells were transfected with one of the TRABID shRNAs for 72 hours, and total RNA was extracted. The levels of Axin2 mRNA were determined by qRT-PCR using beta-actin as control. F,G) Effect of overexpression of TRABID WT or C443S mutant on Wnt reporter gene activity. SW480 (E) or HCT116 (F) cells were cotransfected with WT TRABID or C443S TRABID and TOPFLASH or FOPFLASH. After 24 hrs, luciferase activity was determined. Experiments are performed in triplicates, and data are presented as means ± SD. H) Effect of expression of TRABID WT or C443S mutant on Wnt reporter gene activity in HEK293 cells. Cells were transfected as indicated, and luciferase activity was determined next day.