Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: MicroRNA-21 Knockout Exacerbates Angiotensin II-Induced Thoracic Aortic Aneurysm and Dissection in Mice With Abnormal Transforming Growth Factor-β-SMAD3 Signaling.

doi: 10.1161/ATVBAHA.117.310694

Figure Lengend Snippet: Figure 4. Vascular smooth muscle cells (VSMCs) from S3+/−21−/− mice exhibit reduced canonical transforming growth factor-β (TGF- β) signaling in vivo and in vitro. A, Immunofluorescence staining for SMAD7, p-SMAD2, p-ERK, and p-JNK in vivo 7 d after angio- tensin II (AngII) infusion (n=5–8/group). B and C, Western blotting for canonical and noncanonical TGF-β signaling in primary aortic VSMCs treated with AngII for 24 h in vitro (n=6/group). D, Real-time polymerase chain reaction (RT-PCR) revealed the expression of genes related to TGF-β signaling (Serpine1, Id1, and Ctgf) in primary aortic VSMCs from Smad3+/− and S3+/−21−/− mice after treat- ment with AngII for 24 h (n=8/group). E, RT-PCR showed the expression of genes related to TGF-β signaling (Serpine1, Id1, and Ctgf) after treatment with agomiR-21 or antagomiR-21 in vitro (n=8/group). A 2-tailed Student t test was used for comparisons between 2 groups. The data are presented as the means±SDs for each group. *P<0.05 and **P<0.01. Scale bars: 100 μm (×200). ns indicates nonsignificance.

Article Snippet: The membranes were blotted with primary antibodies against p-SMAD2 (no. 3108, 1:1000), p-ERK1/2 (no. 4370, 1:2000), p-JNK (no. 4668, 1:2000), p-MEK1/2 (no. 9154, 1:1000), T-SMAD2 (no. 3122, 1:1000), T-ERK1/2 (no. 4696, 1:1000), T-JNK (no. 9252, 1:500), MEK1/2 (no. 8727, 1:1000), cyclin D1 (no. 2978, 1:1000), and caspase-3 (no. 9662, 1:1000) from Cell Signaling Technology (MA), SMAD7 (no. MAB2029, 1:250) and MMP9 (no. AF909, 1:2000) from R&D Systems (MN), and PAI-1 (GTX100550, 1:1000) from GeneTex overnight at 4°C.

Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: REPRODUCTION

Article Title: SMAD7 antagonizes key TGFβ superfamily signaling in mouse granulosa cells in vitro

doi: 10.1530/rep-13-0093

Figure Lengend Snippet: Figure 1 Expression and localization of SMAD7 in mouse ovary. (A) Smad7 mRNA was expressed in mouse granulosa cells. RT Neg, no reverse transcriptase. (B, C, D, and E) Immunostaining of SMAD7 using ovaries from untreated immature mice (B) and PMSG (48 h)ChCG (48 h)-treated mice (D) and controls (E). Negative control without primary antibody is depicted in (C). Note that omission of the primary antibody abolished the SMAD7 signals. GC, granulosa cell; CL, corpus luteum. Arrows indicate follicles. Scale barZ50 mm (B and C) and 100 mm (D and E). (F) Gonadotropin administration reduced Smad7 mRNA levels in the ovary. Total RNA was isolated from ovaries of age-matched untreated controls (CTRL; nZ4/group), PMSG (48 h)-treated (nZ4), and PMSG (48 h)/hCG (48 h)-treated mice (nZ3). Gene expression was analyzed by qPCR using the DDCT method. Smad7 mRNA levels in the treatment group were expressed as fold change of the corresponding age-matched controls. Gapdh was used as an internal control. Data represent meansGS.E.M. *P!0.05 vs the corresponding CTRL.

Article Snippet: The sections were then blocked with 3% goat serum for 30 min and incubated with rabbit anti-SMAD7 antibody (Imgenex; 1:300) (Reynolds et al. 2008) at 4 8C overnight.

Techniques: Expressing, Reverse Transcription, Immunostaining, Negative Control, Isolation, Gene Expression, Control

Journal: REPRODUCTION

Article Title: SMAD7 antagonizes key TGFβ superfamily signaling in mouse granulosa cells in vitro

doi: 10.1530/rep-13-0093

Figure Lengend Snippet: Figure 6 Hypothetical model depicting the role of SMAD7 in ovarian granulosa cells. In mouse granulosa cells, TGFb family ligands such as TGFB1, BMP4, and GDF9 are key regulators of granulosa cell function and follicular development. BMP4 and TGFB1/GDF9 signal through ALK2/3/6 and ALK4/5/7 to regulate granulosa cell gene expression and cellular responses/functions. Our data suggest that SMAD7, an inhibitory SMAD, may preferentially inhibit TGFB1/GDF9-induced cumulus expansion-related transcripts in mouse granulosa cells. SMAD7 induced by TGFB1/GDF9 probably acts as a negative feedback to limit TGFB1/GDF9 signaling, although its direct role in mediating gene expression and cellular functions remains to be elucidated.

Article Snippet: The sections were then blocked with 3% goat serum for 30 min and incubated with rabbit anti-SMAD7 antibody (Imgenex; 1:300) (Reynolds et al. 2008) at 4 8C overnight.

Techniques: Cell Function Assay, Gene Expression

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 1. Smad7 expression is induced by hypoxia. A, cDNA microarray comparing 16 h hypoxic (1% O2) and normoxic (21% O2) gene expression in HepG2 cells. B, QRT-PCR study of Smad7 and Glut-1 mRNA in HeLa and HaCaT cell lines under 6 and 16 h of hypoxic or CoCl2 exposure. The hypoxic induction of Smad7 correlates with Glut-1. C, QRT-PCR study of Smad7 in HUVECs demonstrating the activation of Smad7 in hypoxia. D, Oncomine database analysis of Smad7 in coronary artery endothelial cells exposed to hypoxia (2% O2) and anoxia (0% O2; ref. 16).

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Expressing, Microarray, Gene Expression, Quantitative RT-PCR, Activation Assay

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 2. Induction of Smad7 gene is HIF-1α and pVHL-dependent. A, HeLa cells were transfected with empty vector (pcDNA3), HIF-1α or HIF-2α expression plasmids followed by QRT-PCR analysis of Smad7 and Glut-1. The Western blot inset shows expression of HIF-1α and -2α upon the indicated transfection. B, depleting HIF-1α by siRNA abolishes hypoxic induction of Smad7 gene. In nontarget (scrambled) siRNA exposed HeLa cells both Smad7 and Glut-1 were activated by hypoxia. The activation was attenuated by siHIF-1α. Inset; HIF-1α Western blot. C, comparison of Smad7 and Glut-1 expression in pVHL negative (RCC4−) and pVHL rescue transfected (RCC4+) cells under normoxia. D, Oncomine database analysis of studies comparing hypoxia and anoxia in healthy renal cells (16). Example of an analysis extracted from Oncomine database revealed upregulation of Smad7 in RCC (22, 23).

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay, Comparison

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 3. Hypoxic Smad7 upregulation inhibits TGF-β signaling. A, phosphospecific Smad3 (p-Smad3) and total Smad3 (t-Smad3) Western blot analysis demonstrating induction of Smad3 phosphorylation by TGF-β in HaCaT cells under normoxia. In hypoxia, Smad3 phosphorylation is inhibited. B, quantification of p-Smad3 and t-Smad3 levels under the indicated conditions. p-Smad3 levels were correlated to t-Smad3 and t-Smad3 to β-actin. C, Smad3 phosphorylation in HeLa cells exposed to nontarget or two independent Smad7 siRNAs (S7#1 and S7#2). D, HeLa cells exposed to the indicated siRNA followed by exposure to TGF-β and CoCl2. C and D, all samples were run on one gel and the white bars indicate removal of unnecessary information.

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Western Blot, Phospho-proteomics

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 4. Hypoxia activates HaCaT cell invasion in a Smad7-dependent manner. A, invasion of UT-SCC-7 cells compared with HaCaT cells in standard Matrigel invasion assay under normoxia and hypoxia. The high basal invasion capacity of UT-SCC-7 cells was not affected by hypoxia or TGF-β. HaCaT cells with low basal invasion capacity in normoxia become strongly invasive under hypoxia and TGF-β exposure. B, Hoechst-stained HaCaT cells invaded through Matrigel under the indicated conditions. C, quantification of cells invaded through Matrigel under the exposure of the indicated siRNAs in hypoxia (1% O2) and normoxia. Nontarget siRNA transfected HaCaT cells are invasive only under simultaneous TGF-β and hypoxia. The hypoxia-activated HaCaT cell invasion is lost when exposed to either of the Smad7 siRNA (S7#1 and S7#2) or HIF-1α siRNA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Invasion Assay, Staining, Transfection

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 5. Hypoxia reverts the anti-invasive function of Smad7. A, Matrigel invasion assay using adenovirally expressed Smad7 (Ad-Smad7) or LacZ control (Ad-LacZ) for UT-SCC-7 cells under the indicated conditions. Left, Western blot analysis for LacZ and Smad7 expression. Forced Smad7 expression inhibits UT-SCC-7 invasion in normoxia but not in hypoxia (***, P < 0.0001; NS, nonsignificant). B, change (%) in the invasion capacity of UT-SCC-7 cells as compared with normoxic TGF-β–treated and LacZ-infected cells. C, Matrigel invasion assay using Ad-Smad7 or Ad-LacZ exposure in HeLa cells under the indicated conditions (*, P < 0.01; NS, nonsignificant). D, change (%) in the invasion capacity of HeLa cells as compared with normoxic TGF-β–treated and LacZ-infected cells.

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Invasion Assay, Control, Western Blot, Expressing, Infection

Journal: Cancer Research

Article Title: Hypoxic Conversion of SMAD7 Function from an Inhibitor into a Promoter of Cell Invasion

doi: 10.1158/0008-5472.can-09-3777

Figure Lengend Snippet: Figure 6. Smad7 gene is upregulated in HNSCC and associates with tumor hypoxia. A, 28 HNSCC patient samples and 13 anatomically matching normal samples analyzed for Smad7, Smad4, Glut-1, and LDH-A mRNA levels by QRT-PCR. The expression was normalized to EF-1α in each sample. Each dot represents the expression in one sample. For each gene, the P value between control and tumor samples and the median level of expression (line) are shown. B, four independent head and neck studies extracted from the Oncomine database (24–27) show overexpression of Smad7 in cancer tissue. C, Smad7 gene upregulation in HNSCC samples correlates with Glut-1 expression. Spearman correlation coefficient and P-values for the correlation between Smad7, Smad4, and Glut-1 expression. D, Oncomine database–extracted data comparing primary and metastatic tumors (26, 33).

Article Snippet: Western blots were performed in 5% bovine serum albumin in TBS 0.1% Tween 20 with primary antibodies: P-Smad3 (R&D Systems) at 1:2,500, Smad7 (Imgenex, Abcam, Abnova, R&D Systems, or Upstate) at 1:2,500, HIF-1α (Transduction Laboratories) at 1:2,500 and AC-40 actin (SigmaAldrich) at 1:5,000.

Techniques: Quantitative RT-PCR, Expressing, Control, Over Expression

Journal: Molecular therapy. Nucleic acids

Article Title: Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

doi: 10.1016/j.omtn.2024.102174

Figure Lengend Snippet: Figure 6. Smad7 is identified as an essential target of miR-25 in cardiac fibrosis (A) The alignment results between miR-25 and Smad7 30-UTR provide insights into the specific binding locations within the genome sequence. (B) Luciferase reporter activity was measured using Smad7 30UTR in a dose-dependent manner with miR-25 mimic. Additionally, a deletion mutation in the miR-25 binding site of Smad7 30UTR was applied to validate the luciferase activity. Protein levels of SERCA2a and Smad7 were examined by western blot analysis after AAV9 pre-miR-25 delivery in WT mice (C), or lentiviral shSmad7 vector delivery (D). (E) AAV9 miR-25 TuD was injected into mdx/utrn (+/) mice, with or without the lentiviral shSmad7 vector, and protein levels of SERCA2a and Smad7 were detected by western blot analysis. (F) Following euthanasia, mouse hearts were cryosectioned and subjected to Masson’s trichrome staining for the evaluation of cardiac fibrosis. In the western blot analysis, 15 mg of proteins was loaded into each well. For (A)–(F), the data are presented as mean ± SD. *p < 0.05, **p < 0.005 (n = 3 for each group).

Article Snippet: To inhibit Smad7 in vitro and in vivo, a shLentivirus against Smad7 was purchased from OriGene (Cat No. TL514317V; MD, USA).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Mutagenesis, Western Blot, Plasmid Preparation, Injection, Staining

Journal: Molecular therapy. Nucleic acids

Article Title: Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

doi: 10.1016/j.omtn.2024.102174

Figure Lengend Snippet: Figure 7. AAV9 miR-25 TuD reduces fibrosis by inhibiting TGF-b signaling pathways by restoring Smad7 expression Western blotting was performed to evaluate the expres- sion of Smad7 and GAPDH in the canine (GRMD) models (A), with n = 2 for each group. Similarly, in the mdx/utrn (+/) mouse models, western blot analysis was conducted to assess Smad7 and GAPDH expression (B), with n = 5 for each group. (C) The expression levels of pre- and mature miR-25 were quantified using qRT- PCR in mdx/utrn (+/) mice treated with AAV9 control or AAV9 miR-25 TuD, with n = 5 for each group. (D) Furthermore, qRT-PCR analysis was employed to analyze the mRNA expression levels of fibrosis markers, such as fibronectin, galectin-3, and TGF-b1, in comparison with the WT group. In the western blot analysis, 20 mg of proteins was loaded into each well. For (A)–(D), the data represent the mean ± SD. *p < 0.05, **p < 0.005, ***p < 0.001.

Article Snippet: To inhibit Smad7 in vitro and in vivo, a shLentivirus against Smad7 was purchased from OriGene (Cat No. TL514317V; MD, USA).

Techniques: Protein-Protein interactions, Expressing, Western Blot, Quantitative RT-PCR, Control, Comparison