Fig. 5

Epigenetic transcriptional activation of the SMAD3/CBP/P300 complex is LINC01977-dependent and can potentially serve as a target for combination therapy. A RNA-binding abilities of LINC01977 and CBP/P300 were predicted by OmiXcore. B RNA pull-down and western blot assays were used to evaluate the interaction between LINC01977 and CBP/P300. C RIP and qRT-PCR assays were used to measure the enrichment of LINC01977. D Co-immunoprecipitation (co-IP) of CBP or P300 with phosphorylated SMAD3 Ser423/425 in A549 cells transfected with pcDNA3.1 or LINC01977 followed by treatment with TGF-β (10 ng/mL). E Co-IP assays of CBP or P300 with phosphorylated SMAD3 Ser423/425 in A549 cells, which were transfected with LINC01977 followed by treatment with TGF-β (10 ng/mL), and treated with RNase inhibitor or RNase. F Represent images of H3K27ac, assayed by IF confocal microscopy in A549 cells treated with TGF-β (10 ng/mL). Actin, Phalloidin (red); nuclei, DAPI (blue); and H3K27ac (green). Scale bar: 20 μm. (G, H) ChIP assays suggested that LINC01977 SE (G) and promoter (H) were co-occupied by CBP, P300 and H3K27ac. I Abilities of invasion and migration were diminished by combination of LINC01977-ASO and inhibition of CBP/P300, SGC-CBP30 (5 μM) in vitro. J Schematic representation of lung metastasis nude mouse model. K Representative images of IVIS detection and metastatic loci in the lung derived from mouse model. L Mouse overall survival after tail-vein injection. M H&E stained and phosphorylated SMAD3 Ser423/425, CBP, and ZEB1 IHC detection of serial sections from lung metastasis nude mouse model. Representative images are shown. H&E, scale bars: 500 μm; IHC, scale bars: 50 μm. Data in C, G–I are representative of three independent experiments and presented as mean ± S.D., n = 3 biologically independent samples, and the P value was determined by a two-tailed unpaired Student’s t test and one-way ANOVA, respectively. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s. not significantly