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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

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

Fig. 4

LUAD cells adapt to TAM2-induced TGF-β enrichment in the tumor microenvironment via SE-LINC01977-dependent TGF-β/SMAD3 signaling pathway activation. A Western blotting detection of phosphorylated SMAD3 (Ser423/425) levels in A549 cells transfected with pcDNA3.1 or LINC01977 followed by treatment with TGF-β (10 ng/mL) for the indicated times (left panel). Quantitation of protein levels (right panel). B Represent images of phosphorylated SMAD3 (Ser423/425) nuclear translocation, assayed by immunofluorescence confocal microscopy in A549 cells with LINC01977 overexpressed followed by treatment with TGF-β (10 ng/mL) (left panel). Actin, Phalloidin (red); nuclei, DAPI (blue); and phosphorylated SMAD3 Ser423/425 (green). Scale bar: 20 μm. Quantification of nuclear fluorescence of p-SMAD3 relative to total nucleus fluorescence. C Western blotting detection of phosphorylated SMAD3 (Ser423/425) levels in nucleoplasm and cytoplasm fractions of A549 cells transfected with pcDNA3.1 or LINC01977 followed by treatment with TGF-β (10 ng/mL) (left panel) Quantitation of protein levels (right panel). D Western blotting detection of phosphorylated SMAD3 (Ser423/425) levels in nucleoplasm and cytoplasm fractions of A549 cells transfected with Scramble or LINC01977-ASO followed by treatment with TGF-β (10 ng/mL) (left panel). Quantitation of protein levels (right panel). E Confocal images showing the co-localization of LINC01977 and phosphorylated SMAD3 (Ser423/425) in A549 cells transfected with pcDNA3.1 or LINC01977 followed by treatment with TGF-β (10 ng/mL) for 2 h. Scale bar: 2 μm. Fluorescence intensity was quantified using ImageJ. F qRT-PCR detection of LINC01977 and SMAD3 expression in A549 cells treated with scramble or LINC01977-ASO. G qRT-PCR detection of LINC01977 and SMAD3 expression in A549 cells transfected with pcDNA3.1 or LINC01977. H qRT-PCR detection of LINC01977 expression in A549 cells treated with TGF-β (10 ng/mL), TGF-β inhibition SB431542 (10 μM) and SIS3 (5 μM) as indicated. I The LINC01977 promoter contains SMAD3-binding sites. J ChIP assays suggested that SMAD3 bound to the LINC01977 promoter. (K) ChIP assays suggested that SMAD3 bound to the LINC01977 SE. L SMAD3 overexpression elevated the SE-induced transcriptional activity of LINC01977 in A549 cells. M Schematic representation of THP-1-derived M2-like macrophages and co-culture model. N M2 marker expression, CD68 and CD206, were detected by IF staining. Scale bar: 50 μm. (O) Cell proliferation was assessed by EdU incorporation assay; EdU (green); and nuclei, DAPI (blue), respectively (upper panel). Abilities of invasion and migration were detected by transwell assays, respectively (middle and lower panel). Scale bar: 50 μm. Data in A, C, E–K, O 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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