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Fig. 1 | Molecular Cancer

Fig. 1

From: Long noncoding RNA GAS5 inhibits progression of colorectal cancer by interacting with and triggering YAP phosphorylation and degradation and is negatively regulated by the m6A reader YTHDF3

Fig. 1

Screening and identification of YAP-interacting lncRNA GAS5. a RIP-seq experiments were performed to identify YAP-binding lncRNAs. Volcano plot showing the differentially expressed lncRNAs upon YAP immunoprecipitation. Red dots mark top eight upregulated lncRNAs (fold change > 2, FDR < 0.05). b Screening strategy was used to find key YAP-binding lncRNAs in colorectal cancer (CRC). c RIP assays for YAP were performed and the coprecipitated RNA was subjected to qRT-PCR for GAS5 (upper panel). Agarose electrophoresis of PCR products (bottom panel). Experiments were performed in triplicate, and data are presented as mean ± SD. ***P < 0.001. d The expression of YAP and GAS5 were analyzed by qRT-PCR in various CRC cell lines. e GAS5 and YAP expressions in various CRC cell lines, shown by northern blot and western blot (IB). f-g GAS5 facilities YAP cytoplasmic retention as demonstrated by immunofluorescence staining (f) and western blot (g). h RNA pull-down assay (upper panel) and western blot assays (bottom panel) showed that biotinylated-GAS5 could bind with YAP in CRC cells in vitro. i Immunoblot (IB) detection of YAP, which were pulled down by in vitro transcribed biotinylated RNAs corresponding to different fragments of GAS5 in CRC cells. j IB detection of GFP-tagged YAP (WT versus domain truncation mutants) precipitated by in vitro transcribed biotinylated-GAS5 in HEK293T cells. Upper panel: graphic illustration of the domain structure of YAP. k Visualization of interaction between YAP, the 3D structure of which was shown, and GAS5, the secondary structure model of GAS5 was simulated by RNAstructure software. l Bio-layer interferometry (BLI) analysis of biotinylated-GAS5 binding to YAP protein

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