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. 2015 Oct 15;6(35):37257–37268. doi: 10.18632/oncotarget.6133

Figure 2. Wnt2 is required for β-catenin target gene activation in CRC cells.

Figure 2

A. Co-expression of WNT2 with AXIN2 in CRC. Co-expression analysis of WNT2 with AXIN2, using Oncomine database. P value < 0.0001; fold change > 2. B. Depletion of endogenous Wnt2 by shRNAs. Lentiviruses encoding two different shRNAs (#1 and #2) were stably transduced into HCT116 cells. IB assays. Of note, Wnt5A expression was not affected by Wnt2 depletion. C. and D. Downregulation of β-catenin transcriptional activity by Wnt2 KD in CRC cells. HCT116 cells (shGFP or shWnt2) were transiently transfected with pMegaTOPFLASH (TOP) or pMegaFOPFLASH (FOP) β-catenin luciferase reporter plasmids, with pRenilla plasmids (internal control). 48 hours after transfection, cells were analyzed for luciferase activity C.. Other CRC cell lines D.. iCRT14 served as a positive control for inhibition of β-catenin activity. Wnt2-expressing plasmids were co-transfected for rescue assays. E. KO of WNT2 alleles by CRISPR gene targeting system. HCT116 cells were transduced with lentiviruses encoding Cas9 and gRNAs (1 and 2 indicate two different gRNAs). IB analysis. F. Downregulation of β-catenin target genes by WNT2 KO. HCT116 (WNT2 WT vs. WNT2 KO) cells were analyzed by quantitative reverse transcriptase PCR (qRT-PCR) of AXIN2, CD44, and CD133. G. Suppression of β-catenin transcriptional activity by WNT2 KO. β-catenin reporter assays of HCT116 (WNT2 WT vs. WNT2 KO). H. Dephosphorylation of LRP6 by WNT2 KO. IB analysis of HCT116 (WNT2 WT vs. WNT2 KO). I. Upregulation of β-catenin protein by Wnt2. 293T and CCD841CoN cells were transfected with plasmids encoding Wnt2. 24 hours after transfection, cells were analyzed for IB of β-catenin.