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. 2009 Mar 20;37(9):3007–3020. doi: 10.1093/nar/gkp179

Figure 5.

Figure 5.

The activity of the Wnt/β-catenin-dependent reporters positively correlates with the amounts of Dazap2 in the cells. (A) HEK 293 cells containing the integrated reporter pSuperTOPFLASH (STF 293 cells) were transfected with four different Dazap2-specific siRNAs marked 1 to 4 or with control siRNA (EGFP-specific) and the changes in the levels of Dazap2 mRNA or protein were tested 24 h post transfection. The left panel shows the results obtained by qRT–PCR analysis. The relative abundance of Dazap2 mRNA in Dazap2 siRNA- versus control siRNA-transfected cells (the level of Dazap2 mRNA in control cells was set to 100%) was derived from the average CT values after normalizing to the levels of β-ACTIN cDNA. Data from two independent experiments performed in triplicate were combined in the graph. SDs (standard deviations) are shown by error bars. The right panel shows western blots of whole cell extracts prepared from STF 293 cells treated with siRNAs as indicated. Blots were probed with anti-Dazap2, anti-TCF-4 or anti-α-tubulin (as an internal control) antibodies. (B) Results of the reporter gene assays in human cells. STF 293 and DLD-1 adenocarcinoma cells were transfected with Dazap2 or control siRNAs. To stimulate the integrated pSuperTOPFLASH in STF 293 cells the siRNA transfection mixtures additionally contained a Wnt1-expression construct; mixtures including an empty vector were used in control transfections. DLD-1 cells were co-transfected with siRNA and the Tcf/β-catenin-responsive reporter pTOPFLASH or pFOPFLASH as a negative control. After 24 h, cells were harvested and luciferase (firefly) activities were determined in the lysates. The histograms represent mean values of triplicate experiments with SDs. The reporter activity in unstimulated (empty vector) STF 293 cells was arbitrarily set to 1. For DLD-1 cells the average luciferase light units per second (RLU/s) are given; the values were corrected for the efficiency of transfection using the internal control Renilla luciferase expression plasmid. The results of one representative experiment from two in total are shown.