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Fig. 3 | Journal of Hematology & Oncology

Fig. 3

From: Super-enhancer hijacking LINC01977 promotes malignancy of early-stage lung adenocarcinoma addicted to the canonical TGF-β/SMAD3 pathway

Fig. 3

LINC01977 binding to SMAD3 protein is dependent on the MH2 domain. A PCR products from the 3′-RACE and 5′-RACE procedures as shown by agarose gel electrophoresis. B Full length of LINC01977 predicted in the UCSC Genome Browser and measured LINC01977 acquired by 3′-RACE and 5′-RACE. C LINC01977 is predicted to have five stem-loop structures, which were indicated with dashed circles. D Coding potential was determined by Coding Potential Assessment Tool (CPAT). E qRT-PCR detection of LINC01977 expression in the cytoplasmic and nuclear fractions. F Subcellular localization of LINC01977 detected by FISH. Scale bar: 5 μm. G Protein retrieved from the LINC01977 pull-down assay in A549 cells were analyzed by SDS-PAGE. Specific band is indicated with arrow. H Protein retrieved from the LINC01977 pull-down assay in LINC01977 overexpressing A549 cells were analyzed by SDS-PAGE (left panel). Specific bands are indicated with red boxes, and results from LC/MS analysis were shown (right panel). I Schematic representation of RNA pull-down assay. J RIP assays were performed using anti-SMAD2/3/4 antibody in A549 cells. qRT-PCR was used to measure the enrichment of LINC01977. K RNA pull-down assays were performed using in vitro transcribed biotinylated full-length LINC01977 and magnetic beads (as negative control). Western blot was used to evaluate the interaction between LINC01977 and SMAD2/3/4. L Immunoblot detection of the SMAD3 protein in A549 cells as retrieved by in vitro transcribed biotinylated RNAs of different constructs of LINC01977 or its antisense sequence (as negative control). M, (N) RNA pull-down assays were performed using in vitro transcribed biotinylated full-length LINC01977 and its antisense sequence (as negative control), as well as different HA-tagged SMAD3 protein truncations. Western blot was used to evaluate the interaction between LINC01977 and SMAD3 protein truncations by anti-HA antibody. Data in E and J 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

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