Fig 1: SMAD4, a target gene of miR-574-5p, is regulated by MTX2-6. (A) Predicted binding targets for miR-574-5p binding target via miRWalk, miRDB, TargetScan, and miRTarBase. (B) The predicted binding sites between SMAD4 and miR-574-5p. (C) Luciferase reporter assay was conducted to verify that miR-574-5p bound to the 3'-UTR region of SMAD4 directly. miR-574-5p overexpression significantly suppressed, while miR-574-5p loss increased the luciferase activity that carried wild-type (wt) but not mutant (mut) 3'-UTR of SMAD4. (D) RIP assays confirmed the binding status between miR-574-5p and SMAD4 in ESCC cell lines, respectively. (E) qRT-PCR was used to detect the ESCC expression in 72 ESCC tissues and paired adjacent tissues. (F) SMAD4 expression in different tumor size of ESCC. (G,H) Correlation analysis of the expression of MTX2-6, miR-574-5p, and SMAD4 in 80 ESCC samples. (I) Relative mRNA and protein levels of SMAD4 in ESCC cells transfected with miR-574-5p mimics and control groups. (J) Relative mRNA and protein levels of SMAD4 in ESCC cells transfected with LV-MTX2-6 and control groups. (K) Relative mRNA and protein levels of SMAD4 in ESCC cells co-transfected with MTX2-6 + Ctl and MTX2-6 + miR-574-5p. (L) Online Kaplan–Meier overall survival (OS) curves according to SMAD4 expression levels of ESCC. ***p < 0.001.
Fig 2: The regulation of SMAD4 on ESCC cells is mediated by miR-574-5p. (A) Expression of SMAD4 was confirmed by qRT-PCR and western blot in co-transfected ESCC cell lines. (B–E) CCK-8 assays, colony formation assays, and EdU assays were used to detect the cell proliferation after co-transfecting ESCC cells with miR-574-5p + Ctl and miR-574-5p + SMAD4 (scale bar: 100 µm for Edu assay). (F) Western blot analysis confirmed that MTX2-6 inhibits cell cycle process and promotes cell apoptosis through regulating miR-574-5p of ESCC. (G) The IHC of CDK4, Cyclin D1, Caspase3, and Bax in the high expression SMAD4 of tumors and low expression SMAD4 of tumors. (H) Proposed model of lnc MTX2-6 suppresses proliferation by acting as ceRNA of miR-574-5p to modulate SMAD4 in ESCC cells. **p < 0.01, ***p < 0.001.
Fig 3: Restoration of SMAD4 expression abrogated miR-146a-mediated cell migration and invasion. (A and B) The SMAD4 mRNA or protein expression level was detected using western blotting or reverse transcription-quantitative polymerase chain reaction in A375 cells co-transfected with SMAD4 overexpression vector and miR-146a mimics. (C) Transwell assay was performed to detect migration and invasion ability in A375 cells co-transfected with SMAD4 overexpression vector and miR-146a mimics. *P<0.05, **P<0.01 and ***P<0.001 vs. Con+pcDNA3.1 group; #P<0.05 and ##P<0.01 vs. miR-146a mimics+pcDNA3.1 group. SMAD4, Mothers against decapentaplegic homolog 4; miR, microRNA; Con, control.
Fig 4: SMAD4 is required for human cardiomyocyte differentiation. (A): Flow cytometry analysis demonstrates SMAD4 mutant human embryonic stem cells (hESCs) fail to form TNNT2+ cardiomyocytes. Compare left panel hESC with middle and right panel mutant SMAD4 clones C1 and C2, respectively. (B): SMAD4 rescue construct used to overexpress/rescue SMAD4 in wild-type and the mutant clones contains a UbC promoter driving V5-tagged SMAD4. (C): Flow cytometry analysis demonstrates rescued clones recue cardiac differentiation to became TNNT2+ cardiomyocytes. (D): Flow cytometry analysis demonstrates SMAD4 mutated hESCs failed to become TNNT2+ cardiomyocyte using growth factor-based differentiation method. (E): Summary graph of TNNT2 flow cytometry of SMAD4 WT, mutant clones, rescue clones, and growth factor-based differentiation method. *, p < .0001; compared with ES03 (n = 3). #, p < .0001; compared with SMAD4.C1 (n = 4). +, p < .0001; compared with SMAD4.C2 (n = 4). ES03 + Rescue.C2 with similar level SMAD4v5 expression as the mutant rescue clones. Ns, p = .2664 (n = 4). &, p < .0001; compared with ES03.AB (n = 3). AB indicates Activin a/BMP4 differentiation protocol.
Fig 5: SMAD4 mutant fail to form cardiac mesoderm precursor cells and defaults to neuroectoderm fate under conditions for cardiac differentiation. (A): Immunostaining demonstrates expression of the cardiac progenitor marker ISL1, cardiac mesoderm marker MESP1 and cardiomyocyte marker NKX2-5 after 3 days cardiac differentiation. Scale bar = 100 µm. (B): Immunostaining demonstrates expression of the pluripotent marker POU5F1, cardiac marker NKX2-5, and neuroectoderm marker PAX6 after 6 days cardiac differentiation. Scale bar = 100 µm.
Supplier Page from Abcam for Anti-Smad4 antibody