Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Phenazopyridine promotes RPS23RG1/Rps23rg1 transcription and ameliorates Alzheimer-associated phenotypes in mice.

doi: 10.1038/s41386-022-01373-7

Figure Lengend Snippet: Fig. 5 SMAD3 binds to the promoter of RPS23RG1 and regulates its expression. A The pGL3-basic reporter vectors containing progressive 3’ deletions of the RPS23RG1 promoter were transiently transfected into SH-SY5Y for 24 h. The pRL reporter plasmid was co-transfected as an internal control to correct the transfection efficiency. One-way ANOVA; ***p < 0.001 for comparisons to the control (pGL3) group; ###p < 0.001 for comparisons to the −1509/+1187 group; n = 3. B A putative SMAD3 binding site was found at the +1180 to +1184 positions of the RPS23RG1 promoter. C ChIP assays were used to pulled down SMAD3 bound genomic DNAs using an antibody against SMAD3 or normal IgG (as a negative control). PCR was performed using immunoprecipitated DNAs or input DNAs as templates. PCR products were separated on a 2% agarose gel. D The +732/+1187 cell line was treated with SIS3 (10 μM) for 12 h. Then luciferase activity was quantified for comparison. Student’s t test; ***p < 0.001; n = 3. E SH-SY5Y cells were treated with SIS3 (10 μM) for 12 h. Endogenous RPS23RG1 mRNA levels were determined. Student’s t test; **p < 0.01; n = 3. F SH-SY5Y and N2a cells were treated with phenazopyridine and SIS3 individually or collectively for 12 h. Human RPS23RG1 and mouse Rps23rg1 mRNA levels were determined by quantitative real-time PCR for comparison. One-way ANOVA test; *p < 0.05, ns not significant; n = 3. G SH-SY5Y cells were treated with phenazopyridine for 12 h. ChIP assays were performed using an antibody against SMAD3. PCR was performed using immunoprecipitated DNAs or input DNAs as templates. PCR products were separated on a 2% agarose gel. PCR band intensities were quantified and normalized to those of inputs for comparison. **p < 0.01; n = 3. H N2a cells were treated with phenazopyridine for 12 h and then subjected to western blot analysis to determine RPS23RG1 and p-SMAD3 levels. Actin was used as a loading control. I SH-SY5Y cells were treated with phenazopyridine for 12 h. The localization of SMAD3 (in red) was detected by immunofluorescence. DAPI was used to stain the nuclei (in blue). Scale bars: 20 μm.

Article Snippet: Phenazopyridine and the SMAD3 inhibitor SIS3 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Binding Assay, Negative Control, Immunoprecipitation, Agarose Gel Electrophoresis, Luciferase, Activity Assay, Comparison, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: Respiratory Research

Article Title: The canonical TGF-β/Smad signalling pathway is involved in PD-L1-induced primary resistance to EGFR-TKIs in EGFR-mutant non-small-cell lung cancer

doi: 10.1186/s12931-019-1137-4

Figure Lengend Snippet: PD-L1 induced EMT by upregulating Smad3 phosphorylation. a Western blot analysis of p-Smad3 and Smad3 in stable PD-L1-overexpressing or PD-L1-silenced HCC827 cells. The PD-L1 blot is the same blot shown in Fig. a and b. b After serum starvation for 24 h, PD-L1-overexpressing and PD-L1-silenced HCC827 cells were treated with SIS3(3 μM) or TGF-β1 (10 ng/ml) for 24 h. The expression of p-Smad3, Smad3, E-cadherin and vimentin was analysed by western blotting. c Knockdown of PD-L1 moderated TGF-β1-induced PAI-1 promoter activation. The relative PAI-1 promoter activities of PD-L1-silenced HCC827 cell lines were measured by a Dual-Luciferase Reporter Assay kit. Relative luciferase activity was expressed as the mean fold change from the basal level ± SD of three independent experiments. d and ( e ) Stable PD-L1-overexpressing and PD-L1-silenced HCC827 cells were treated with or without SIS3(3 μM) and TGF-β (10 ng/ml) for 24 h, respectively, and allowed to migrate through 8-μM pore Transwell inserts. The migrated cells were stained and counted in at least three microscopic fields (magnification, 100×). The cells were allowed to invade through Matrigel-coated Transwell membranes, and invasive cells were stained and counted under a light microscope

Article Snippet: Gefitinib and SIS3 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Knockdown, Activation Assay, Luciferase, Reporter Assay, Activity Assay, Staining, Light Microscopy

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet:

Article Snippet: SIS3 , MedChemExpress , Cat# HY-13013.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Plasmid Preparation, Extraction, Microarray, Software

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 reduces TGF-β 1 -induced EMT in HAT II cells partly through the SMAD and PI3K/AKT signaling pathways. HAT II cells were pre-incubated with 10 μM Sis3 (a specific Smad3 inhibitor), and 10 μM LY294002(PI3Kinhibitor) for 30 min prior to TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - h : Sis3 and LY294002 treatment abolished the inhibition of LXA4 on the EMT in AT II cells. i - k : LXA4 inhibited TGF-β 1 -induced the phosphorylation of AKT and Smad in primary AT II cells. n = 4 for each culture condition, repeated using cells from 4 donors. Data were presented with means ±SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Inhibitors were used at the following concentrations according to manufacturers’ instructions: LY294002, a PI3-kinase inhibitor (Calbiochem, Nottingham, UK); Sis3 (smad3 inhibitor), Boc-2 (N-t-Boc-Phe-Leu-Phe-Leu-Phe; GenScript USA Inc., the ALXR antagonist) and BML-111(Enzo Life Sciences, NY, United States, the ALXR agonist), all at 10 μM.

Techniques: Protein-Protein interactions, Incubation, Inhibition, Phospho-proteomics