Fig 2: Identification of VSNL1 as a novel Wnt/β‐catenin target gene in CRC cells. (A) Strategy for the identification of candidate target genes of the Wnt/β‐catenin signaling pathway. (B) Protein expression levels of VSNL1 and β‐catenin, and mutation statuses of APC and CTNNB1, in six CRC cell lines. (C) Relative expression levels of VSNL1 mRNA in CRC cell lines (HCT116, SW48, and SW480) treated with control siRNA (siCtrl) and two CTNNB1 siRNAs (siCTNNB1 #9 and #10) for 48 h. Data are presented as means ± SEs of ≥3 independent experiments. (D) Protein expression levels of VSNL1 and β‐catenin in CRC cell lines (HCT116, SW48, and SW480) treated with control siRNA (siCtrl) and two CTNNB1 siRNAs (siCTNNB1 #9 and #10) for 48 h.

Fig 3: Induction of apoptosis by VSNL1 silencing in CRC cells. (A) SubG1 population of SW48 and SW480 cells treated with control siRNA or two VSNL1 siRNAs (siVSNL1 #2 and #3). Data are presented as means ± SEs of ≥3 independent experiments. (B) Protein expression levels of cleaved PARP and cleaved caspase‐3 in SW48 and SW480 treated with control siRNA or two different VSNL1 siRNAs (siVSNL1 #2 and #3). (B) Analysis of cleavage of PARP and caspase‐3, ‐8, and ‐9 in SW48 and SW480 cells treated with control siRNA or two VSNL1 siRNAs (siVSNL1 #2 and #3). Band intensities were quantified using ImageJ and were normalized to β‐actin.

Fig 4: Regulation of VSNL1 expression via TCF7L2‐binding elements in intron 1. (A) Genomic structure of VSNL1 and the candidate TCF7L2‐binding elements in intron 1. (B) β‐catenin‐dependent transcriptional activity of the candidate enhancer region (CER). A reporter assay was performed using a reporter plasmid containing the CER (pGL4.23‐CER) or an empty vector (pGL4.23‐EV) in combination with the control siRNA or CTNNB1 siRNA in HCT116 and SW480 cells. (C) TCF7L2‐dependent transcriptional activity of the CER. A reporter assay was performed using the reporter plasmids that contained the CER or empty vector in combination with a dominant‐negative TCF7L2 expression vector (dnTCF7L2) or an empty vector (Mock) in HCT116 and SW480 cells. (D) Interaction of the candidate region with TCF7L2 was analyzed by ChIP‐qPCR with anti‐TCF7L2 antibody. A TCF7L2‐binding region in RNF43 was used as the positive control. (E) Schematic representation of the CER and mutant constructs of VSNL1. (F) Involvement of the three putative TCF7L2‐binding elements in the region with transcriptional activity. A reporter assay was performed using reporter plasmids with mutations in the putative TCF7L2‐binding elements. Data are presented as means ± SEs of ≥3 independent experiments.