Fig. 5.

LINC00265 increases β-catenin signaling via ZMIZ2. a Gene ontology (GO) analysis of the significantly altered genes associated with LINC00265 in the TCGA dataset. b Heatmap shows the relative RNA expression of β-catenin target genes that LINC00265 is positively associated with CRCs from TCGA dataset. c Western blot indicated the β-catenin expression in LINC00265 depletion HCT116 and SW480 cells. d, e The protein level of β-catenin and ZMIZ2 in the indicated stable cells. f, g Depletion of LINC00265 reduced the interaction between ZMIZ2 and β-catenin. HCT116 cells were cotransfected with indicated vectors. IP and IB with indicated antibodies were performed. h ZMIZ2 affects β-catenin protein turnover. HCT116 cells were transfected with indicated vectors. After cells were treated with CHX (50 μg/ml) for the indicated time, expression of β-catenin and ZMIZ2 was analyzed by western blotting (left); the intensity of β-catenin expression for each time point was quantified by densitometry with β-actin as a normalizer (right). i, j Ectopic ZMIZ2 expression increases β-catenin deubiquitination (i) and depletion of ZMIZ2 decreased β-catenin deubiquitination (j). HEK293 cells were transfected with Myc-ubiquitin (Ub), HA-β-catenin, Flag-ZMIZ2 or ZMIZ2 shRNAs. IP and IB were performed with the indicated antibodies. k ZMIZ2 promotes K6-, K33- and K48-linked deubiquitination of β-catenin. HEK293 cells were cotransfected with Flag-β-catenin, ZMIZ2 and HA-Ub or its mutants as indicated