Journal: International Journal of Medical Sciences

Article Title: Kartogenin regulates hair growth and hair cycling transition

doi: 10.7150/ijms.68434

Figure Lengend Snippet: Sequences of primers used for RT-PCR

Article Snippet: Primary antibodies were used as follows: rabbit polyclonal anti-TGF-β2 (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-phosphorylated Smad2 (p-Smad2)/phosphorylated Smad3 (p-Smad3) (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Sequencing

Journal: International Journal of Medical Sciences

Article Title: Kartogenin regulates hair growth and hair cycling transition

doi: 10.7150/ijms.68434

Figure Lengend Snippet: Kartogenin reduced the mRNA and protein levels of TGF-β2/Smad signaling molecules. a-c. Relative mRNA expression levels of TGF-β2, Smad2, and Smad3 in human ORSCs, determined by RT-PCR. d. TGF-β2, p-Smad,2 and p-Smad3 protein expression levels in human ORSCs, detected by western blotting. The grouping of blots was cropped from different gels. e-g. Quantitative analysis of TGF-β2, p-Smad2, and p-Smad3 protein levels. h-j TGF-β2 shown in green, p-Smad2, and p-Smad3 shown in red and nuclei counterstained with DAPI (blue). Merged images indicate the expression and location of TGF-β2, p-Smad2, and p-Smad3.*P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies were used as follows: rabbit polyclonal anti-TGF-β2 (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-phosphorylated Smad2 (p-Smad2)/phosphorylated Smad3 (p-Smad3) (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Respiratory Research

Article Title: Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

doi: 10.1186/1465-9921-11-21

Figure Lengend Snippet: Regulation of TGF-β1-induced phosphorylation of Smad2 and Smad3 by PPARγ ligands . Cell lysates were prepared from A549 cells pre-treated with vehicle, RGZ (10 μM) or CGZ (10 μM) for 1 hr and then treated with TGF-β1 at the indicated concentrations for 1 hr. The Western blot is representative of 3 separate experiments.

Article Snippet: Rabbit monoclonal antibodies against human Smad2 and Smad3, and phosphorylated Smad2 and Smad3 were from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Notch Signaling Activation Suppresses v-Src-Induced Transformation of Neural Cells by Restoring TGF-β-Mediated Differentiation

doi: 10.1371/journal.pone.0013572

Figure Lengend Snippet: (A) Media were collected after a 30-hour incubation of QNR/v-src ts (v-src ts medium) and QNR/v-src ts /ICN (ICN medium) cells at 37°C or 41°C. QNR/v-src ts cells were incubated at 37°C in normal medium (BME) or conditioned media for 1 hour, before lysis. Phosphorylated Smad2 was detected by western-blot. β–actin was used for protein level normalization. (B) QNR/v-src ts cells were treated at 37°C with increasing concentrations of SB431542 inhibitor for 2 hrs and then incubated in ICN medium during 1 hour in presence of inhibitor. Effects of this treatment on Smad2 phosphorylation were analyzed by western-blot. (C) Comparison of mature TGF-β3 levels in v-Src and ICN media. Cells were plated at 2.10 6 cells per 100 mm dish at 37°C or 41°C. One day after plating, growth media were replaced with serum-free BME. After a 30-hour incubation, media were collected and concentrated 50 times using Vivaspin6 columns. 20 µg of proteins were migrated under non-denaturing conditions. Mature TGF-β3 migrates as 25 kDa band. (D) QNR/v-src ts cells were treated, one hour before lysis, with ICN medium preincubated at 37°C for one hour with increasing amounts of neutralizing antibodies directed against TGF-β3. Levels of Smad2 phosphorylation were analyzed by western-blot. Erk was used to normalize protein loading. (E) Levels of phosphorylated Smad2 in QNR/v-src ts /ICN cells treated at 37°C or 41°C with DMSO or 10 µM of SB431542 inhibitor for 24 hrs.

Article Snippet: Blots were probed with primary antibodies used at the following dilutions: anti-phosphorylated Smad2 (Ser465/467; #3101) and anti-phosphorylated MLC2 (Ser19; #367) polyclonal antibodies (Cell Signaling Technology, Hertfordshire, UK) at 1/1000 e ; anti-TGF-β3 monoclonal antibody (MAB243; R&D systems, Minneapolis, MN, USA) at 1/500 e ; anti-Glutamine Synthetase monoclonal antibody (Chemicon, Hampshire, UK) at 1/1000 e ; anti-Erk1 polyclonal antibody (SC-93, Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) at 1/2000 e ; and anti β-actin monoclonal antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) at 1/10000 e .

Techniques: Incubation, Lysis, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

doi: 10.7150/ijbs.69373

Figure Lengend Snippet: Smad7 was the target of miR-520h and regulated the EMT. (A) Venn diagram of the intersection among target genes by miRmap, Starbase, TargetScan and miRDB. (B) RT-qPCR assay demonstrated that nitroxoline led to increased smad7 expression. (C) Western-blotting assay demonstrated that nitroxoline up-regulated the expression of smad7, contributing to the repression of smad2/3 and EMT. (D) Dual luciferase assay showed the direct binding of miR-520h and 3'UTR of smad7 mRNA. (E) The exogenous dysregulation of miR-520h influence the expression of smad7, smad2/3 and EMT markers. (F and G) RT-qPCR and western blotting assay indicated the inhibition of smad7 after the transfection of smad7-si. (H and I) The capacity of migration was rescued by the decrease of smad7, which was inhibited by miR-520h-inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: The following antibodies, monoclonal anti-GAPDH (Catalogue #5174, 1:1000), monoclonal anti-E-cadherin (Catalogue #3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal smad2/3 (Catalogue#8685T, 1:1000) were purchased from Cell signaling Technology; monoclonal anti-EGR1 (Catalogue#55160, 1:1000) was purchased from abcam; monoclonal anti-samd7 (Catalogue#66478-1-lg, 1:1000) was purchased from proteintech; monoclonal smad2/3-phosphorylation (Catalogue#abs130992, 1:1000) were purchased from Absin; anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell signaling Technology.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Binding Assay, Inhibition, Transfection, Migration

Journal: International Journal of Biological Sciences

Article Title: Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

doi: 10.7150/ijbs.69373

Figure Lengend Snippet: CircNDRG1 regulated smad7 expression and inhibited metastasis by targeting miR-520h. (A and B) Transwell and wound-healing assay demonstrated nitroxoline could inhibit the migration of bladder cancer cells, circNDRG1-si further promoted the migration, which was significantly rescued by preventing the expression of miR-520h. (C) Western-blotting assay showed that alteration of the expression levels of smad7, smad2/3 and the EMT-associated markers E-cadherin and N-cadherin, caused by nitroxoline plus circNDRG1-si treatment, were rescued by transfection with the miR-520h inhibitor. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: The following antibodies, monoclonal anti-GAPDH (Catalogue #5174, 1:1000), monoclonal anti-E-cadherin (Catalogue #3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal smad2/3 (Catalogue#8685T, 1:1000) were purchased from Cell signaling Technology; monoclonal anti-EGR1 (Catalogue#55160, 1:1000) was purchased from abcam; monoclonal anti-samd7 (Catalogue#66478-1-lg, 1:1000) was purchased from proteintech; monoclonal smad2/3-phosphorylation (Catalogue#abs130992, 1:1000) were purchased from Absin; anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell signaling Technology.

Techniques: Expressing, Wound Healing Assay, Migration, Western Blot, Transfection