Figure 2

Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription. Panel A, Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B, cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p < 0.001; ***, p < 0.005). Cell extracts also were analyzed for abundance of PP2A C-subunit by immunoblotting. Immunoblots were stained with anti-GAPDH antibodies to establish loading equivalence.Panel C, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1, Super8xTOPFlash (M50) and small t antigen then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and the transcriptional response displayed relative to the unstimulated cells (set to "1"). Cell lysates were analyzed by immunoblotting, blots stained with anti-PP2A C-subunit, anti-GAPDH, or anti-small t antigen antibodies. The results shown are mean values ± S.E. from 5 independent experiments.