Journal: iScience

Article Title: Long noncoding RNA Lnc-DIF inhibits bone formation by sequestering miR-489-3p

doi: 10.1016/j.isci.2022.103949

Figure Lengend Snippet:

Article Snippet: Ser465/Ser467 phosphorylated SMAD2 Rabbit polyclonal antibody , Bioss , Cat# bs-3419R, RRID: AB_10880886.

Techniques: Luciferase, Plasmid Preparation, Recombinant, In Vivo, Transfection, Reporter Assay, In Situ Hybridization, Sequencing, Software

Journal: Journal of experimental & clinical cancer research : CR

Article Title: N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

doi: 10.1186/s13046-024-03006-x

Figure Lengend Snippet: Fig. 4 CircSLCO1B3 regulates miR-502-5p/HOXC8/SMAD3 axis to promote proliferation, migration and invasion in ICC. a Schematic illustration exhibited overlapping of the target mRNAs of miR-502-5p predicted by miRDB, miRTarBase and TargetScan databases. b qPCR was conducted to determine the relative expression levels of 12 potential target mRNAs after downregulation of circSLCO1B3 or miR-502-5p and upregulation of miR-502-5p in ICC cells. c Western blotting was performed to determine the expression of HOXC8 in ICC cells after overexpression or downregulation of circSLCO1B3. d FISH scores statistical analysis of circSLCO1B3 in ICC tissues and normal tissues. e The correlation of HOXC8 and circSLCO1B3 in FISH and IF scores. f, g qPCR showed that the expression of HOXC8 in ICC cell lines and tissues. h CCK-8 assays and trans-well assays were performed to determine the ability of proliferation and migration in ICC cells transfected with HOXC8 vectors and HOXC8 siRNA (magnification, × 200, scale bar, 100 μm). i Schematic illustration and the relative luciferase activities of HOXC8-WT and HOXC8-Mut luciferase reporter vectors. j RNA pull-down assay was executed in HuCCT1 cells to detect the enrichment of HOXC8 by the miR-502-5p probe. k Western blotting was performed to determine the expression of smad2, p-smad2, smad3, p-smad3 in HuCCT1 cells after depleting circSLCO1B3. l Luciferase plasmids with the first 2,200 nucleotides of SMAD3 promoter domain or with blank plasmids were formed to detect the associative relation of SMAD3 and HOXC8. m ChIP assays were executed to detect the relation of SMAD3 promoter domain and HOXC8. n qPCR was conducted to detect the expression relation of HOXC8 and SMAD3 in 13 pairs ICC tissues and normal tissues. Data were showed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Specific primary antibodies for the proteins HOXC8 (1:1000, #15448-1-AP, proteintech), SMAD3(1:1000, #9523, CST), SMAD2(1:1000, 12570-1-AP, proteintech), p-SMAD2 (1:1000, Ser465/467, CST), p-SMAD3 (1:1000, Ser423/425, CST), E-Cadherin (1:1000, 20874-1-AP, proteintech), N-Cadherin (1:1000, 22018-1-AP, proteintech), VIM (1:1000, #5741, CST), PD-L1 (1:1000, AB205921, Abcam) and GAPDH (1:10,000, Abcam, USA) were used to incubate the membranes overnight at 4 °C.

Techniques: Migration, Expressing, Western Blot, Over Expression, CCK-8 Assay, Transfection, Luciferase, Pull Down Assay

Journal:

Article Title: Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad *

doi: 10.1074/jbc.M409486200

Figure Lengend Snippet: TSA201 cells were cotransfected with 50 ng/well of indicated expression vectors (pRK, pRK-Smad2, pRK-Smad4, pcDNA, pcDNA-A3FOXO1) and 500 ng/well of reporter plasmids (cyclin D2 promoter-LUC, epiregulin promoter-LUC, or inhibin-promoter-LUC) using calcium phosphate precipitation, as described under “Experimental Procedures.” Cells were harvested 48 h after transfection and assayed for luciferase activity. Relative light units (RLU) were calculated as a percent inhibition or induction over control group. Values represent mean ± S.E. of three independent experiments. Each separate experiment is replicated in triplicate.

Article Snippet: The following were purchased: anti-phospho-ERK (Thr 202 /Tyr 204 ), anti-phospho-AKT (Ser 473 ), anti-phospho-FOXO1 (Ser 256 ), anti-phospho-Smad2 (Ser 465 /Ser 467 ; also reacts with phospho-Smad3, Ser 423 /Ser 425 ), anti-AKT, and secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies, Beverly, MA); anti-SF-1 (ABR Affinity Bioreagents, Golden, CO); anti-PCNA (Upstate Biotechnology, Lake Placid, NY); anti-cyclin D2, anti-p27, anti-FOXO1, anti-CREB binding protein (CBP), and anti-Gα s (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Smad2 and anti-Smad3 (Zymed Laboratories Inc. South San Francisco, CA).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Inhibition

Journal:

Article Title: Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad *

doi: 10.1074/jbc.M409486200

Figure Lengend Snippet: Model shows that FSH signals to activate PI 3-kinase to promote Akt phosphorylation and consequently phosphorylation of FOXO1, resulting in release from promoters of cyclin D2 and, potentially, epiregulin, aromatase, and SF-1, and exit of phosphorylated FOXO1 from the nucleus. Also necessary for GC proliferation and synergistic activation of differentiation genes is positive signaling through Smad2/3 via activin or a related TGFβ ligand, resulting in transactivation of indicated target genes.

Article Snippet: The following were purchased: anti-phospho-ERK (Thr 202 /Tyr 204 ), anti-phospho-AKT (Ser 473 ), anti-phospho-FOXO1 (Ser 256 ), anti-phospho-Smad2 (Ser 465 /Ser 467 ; also reacts with phospho-Smad3, Ser 423 /Ser 425 ), anti-AKT, and secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies, Beverly, MA); anti-SF-1 (ABR Affinity Bioreagents, Golden, CO); anti-PCNA (Upstate Biotechnology, Lake Placid, NY); anti-cyclin D2, anti-p27, anti-FOXO1, anti-CREB binding protein (CBP), and anti-Gα s (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Smad2 and anti-Smad3 (Zymed Laboratories Inc. South San Francisco, CA).

Techniques: Activation Assay

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: Sequences of the primers for q-PCR.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques:

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: (A) Area semi-quantitative statistical analysis by immunohistochemistry; (B-E)Expression of TGF- β 1, smad2, smad3,col1a1 col3a1and α-SMA by immunohistochemistry. All data are presented as mean ± SD. n = 6 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001; between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Immunohistochemistry, Expressing

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: Quantitative real time-PCR was used to measure the mRNA levels of(A)TGF- β1 ;(B)α-SMA;(C)Smad2;(D)Smad3. All data are presented as mean ± SD. n = 4 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: (A and D) Immunofluorescence was used to measure the protein expression levels of(B)TGF- β 1;(C)Smad3;(E) α -SMA;(F)Smad2. All data are presented as mean ± SD. n = 3 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Immunofluorescence, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Adenovirus-mediated knockdown of activin A receptor type 2A attenuates immune-induced hepatic fibrosis in mice and inhibits interleukin-17-induced activation of primary hepatic stellate cells

doi: 10.3892/ijmm.2018.3600

Figure Lengend Snippet: Knockdown of ACVR2A suppresses Con A-induced activation of hepatic stellate cells in vivo . (A) Relative mRNA and (B) protein expression levels of ACTA2, COL1A1 and COL4A1 were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. (C) Immunohistochemistry was performed to detect the protein expression levels of α-SMA, collagen I and IV. Orange arrowheads, representative α-SMA-positive cells; pink arrowheads, representative collagen I-positive cells; green arrowheads, representative collagen IV-positive cells. Scale bars, 50 μ m. (D) Protein expression levels of p-Smad2 and total Smad2 in liver tissues were detected using western blot analysis. Data are expressed as the means ± standard error (n=5/group). α-SMA, α-smooth muscle actin; ACVR2A, activin A receptor type 2A; Con A, concanavalin A; ; NC, negative control; p-, phosphorylated; shRNA, short hairpin RNA.

Article Snippet: Rabbit polyclonal antibodies against ACVR2A (ab135634, 1:500; Abcam, Cambridge, UK), type I α collagen (collagen I, BA0325, 1:400; Wuhan Boster Biological Technology, Ltd., Wuhan, China), type IV α collagen (collagen IV, BA2174, 1:400; Wuhan Boster Biological Technology, Ltd.), Smad2 (bs-0718R, 1:500), phosphorylated (p)-Smad2 S465/467 (bs-3419R, 1:500; both from BIOSS, Beijing, China) and a mouse monoclonal antibody against α-smooth muscle actin (α-SMA, BM0002, 1:400; Wuhan Boster Biological Technology, Ltd.) were used to detect the expression of corresponding proteins.

Techniques: Activation Assay, In Vivo, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Negative Control, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Adenovirus-mediated knockdown of activin A receptor type 2A attenuates immune-induced hepatic fibrosis in mice and inhibits interleukin-17-induced activation of primary hepatic stellate cells

doi: 10.3892/ijmm.2018.3600

Figure Lengend Snippet: Ad-ACVR2A shRNA suppresses IL-17-induced activation of primary mHSCs in vitro . The primary mHSCs were infected with Ad-ACVR2A shRNA or Ad-NC shRNA for 24 h, and were then stimulated with recombinant IL-17A or IL-17F for an additional 48 h. (A) Knockdown efficiency of Ad-ACVR2A shRNA was confirmed in normal mHSCs by western blot analysis. (B and D) Production of collagens I and VI in cell supernatants were determined by ELISA kits. (C and E) Protein expression levels of α-SMA, p-Smad2 and total Smad2 were determined by western blot analysis. Data are expressed as the means ± standard deviation (n=3/group). α-SMA, α-smooth muscle actin; ACVR2A, activin A receptor type 2A; IL-17, interleukin-17; mHSCs, mouse hepatic stellate cells; NC, negative control; p, phosphorylated; shRNA, short hairpin RNA.

Article Snippet: Rabbit polyclonal antibodies against ACVR2A (ab135634, 1:500; Abcam, Cambridge, UK), type I α collagen (collagen I, BA0325, 1:400; Wuhan Boster Biological Technology, Ltd., Wuhan, China), type IV α collagen (collagen IV, BA2174, 1:400; Wuhan Boster Biological Technology, Ltd.), Smad2 (bs-0718R, 1:500), phosphorylated (p)-Smad2 S465/467 (bs-3419R, 1:500; both from BIOSS, Beijing, China) and a mouse monoclonal antibody against α-smooth muscle actin (α-SMA, BM0002, 1:400; Wuhan Boster Biological Technology, Ltd.) were used to detect the expression of corresponding proteins.

Techniques: shRNA, Activation Assay, In Vitro, Infection, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Negative Control

Journal: Oncology reports

Article Title: Rhapontigenin inhibits TGF-β-mediated epithelial‑mesenchymal transition via the PI3K/AKT/mTOR pathway and is not associated with HIF-1α degradation.

doi: 10.3892/or.2016.4664

Figure Lengend Snippet: Figure 1. A549 cells treated with TGF-β undergo EMT. (A) TGF-β treatment for 24 h led to fibronectin upregulation (A, upper panel) and E-cadherin downregulation (A, lower panel). (B) TGF-β treatment for 24 h led to upregulation of FN and N-cadherin expression, as well as phosphorylation of Smad2, in a dose-dependent manner. (C and D) TGF-β administration for 5 days led to upregulation of FN expression and fibroblastic morphology (C, phase contrast microscope images; D, western blotting data of the upregulation of HIF-1α and FN expression by TGF-β. (E and F) TGF-β administration for 2 or 3 days led to upregulation of FN and HIF-1α in 769-P cells (E). Cells grown on coverslips were treated with 5 nM TGF-β for the indicated time. After fixing the cells in 3% paraformaldehyde and 2% sucrose in PBS, the cells were incubated with rabbit anti-fibronectin antibodies and Alexa 594-labeled anti-rabbit secondary antibodies, mounted on glass slides with mounting medium containing Hoechst 33342, and observed using a fluorescence microscope (F).

Article Snippet: Antibodies against mTOR, mTOR-Ser2448, Akt-Ser473, Akt, S6K-Thr389, S6-Ser235/236, GSK-3β, GSK3β-Ser9, Smad2-Ser465/467, active β-catenin, and Snail were obtained from Cell Signaling Technology.

Techniques: Expressing, Phospho-proteomics, Microscopy, Western Blot, Incubation, Labeling, Fluorescence