Fig. 2

GAS5 facilitates YAP cytoplasmic retention and promotes phosphorylation and ubiquitination-mediated degradation of YAP. a HEK293T cells were co-transfected with the YAP and GAS5 plasmid or indicated siRNA, and the interaction between YAP and LATS1/14–3-3/TEAD1 were studied by immunoprecipitation and western blot. b Western blot showed total and phosphorylated protein of YAP and CTGF, a target gene of YAP in LOVO cells expressing GAS5-specific siRNA or SW620 cells expressing exogenous GAS5. c Western blot showed co-transfected with the YAP and GAS5 plasmid suppressed protein expression of CTGF, a target gene of YAP. d SW620 cells were transfected with YAP plasmids or co-transfected with GAS5 and subjected to a cycloheximide (CHX) chase assay. Immunoblot detection of YAP (left); IB data were quantified using the ImageJ software (right). After 24 h, CHX (10 μg/ml) was added to the cell culture medium, and incubation was continued for 0, 4, 8, 12, or 24 h. Error bars indicate the mean ± SD. ***P <0.001. e Ubiquitination assays of CRC cells co-transfected YAP with GAS5 plasmid (left) or GAS5-specific siRNA (right). The bottom panels depict the input of the cell lysates. Twenty-four hours after transfection, 10 nM MG132 was added to the 1640 culture medium and incubation was continued for 8 h. f SW620 cells were transfected with full length of GAS5 or each of the GAS5 mutants and analyzed with immunoblot. g The co-transfection of His-tagged YAP and exogenous GAS5 or each of the GAS5 mutants was immunoprecipitated with anti-His antibody and analyzed with immunoblot for ubiquitination. h Venn diagram and heatmap of the common differentially expressed genes after overexpression of GAS5 and YAP knockdown are shown. i Intersection analysis of KEGG pathway for differentially expressed genes in YAP knockdown and GAS5 upregulation groups (fold change ≥2; p-value < 0.05 in RNA-seq). j qRT-PCR analysis of cells with exogenous GAS5 or transfected with GAS5-specific siRNA compared with vector controls. Experiments were performed in triplicate, and data are presented as the mean ± SD. **P <0.01; and ***P <0.001