anti smad3  (Cell Signaling Technology Inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FOS drives podocyte injury and renal fibrosis in diabetic kidney disease via direct Smad3 binding

doi: 10.1007/s00018-026-06215-z

Figure Lengend Snippet: shRNA-mediated FOS suppression demonstrated renoprotective effects in DKD rats. ( A , B ) Longitudinal monitoring of physiological parameters: ( A ) body weight and ( B ) blood glucose levels measured fortnightly. ( C – F ) Renal function parameters at study termination: ( C ) urinary microalbumin, ( D ) UACR, ( E ) Scr, and ( F ) BUN. (G–I) Histopathological evaluation of kidney tissues: ( G ) HE staining, ( H ) MASSON staining, and ( I ) PAS staining. Black arrowheads indicate pathological features quantified as glomerulosclerosis. Images acquired at 200× and 400× magnification. ( J ) Immunohistochemical staining of FOS expression in kidney tissues (200× and 800×). ( K ) TUNEL staining for apoptosis detection (200× and 400×). ( L ) Quantification of glomerulosclerosis index from PAS staining. ( M ) Quantification of interstitial fibrosis score from MASSON staining. ( N ) Apoptotic rate quantified as percentage of TUNEL‑positive nuclei in glomerular and tubular regions. ( O ) Percentage of FOS‑positive nuclei determined from IHC images. ( P ) Western blot analysis of FOS, p‑Smad3, Smad3, EMT‑related proteins, and fibrosis‑related proteins. Data in C–F, L–O are presented as mean ± SEM; n = 6 rats per group. Five images were analyzed per biological replicate, with three biological replicates assessed per condition. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Gene-specific siRNAs targeting FOS and Smad3 (Hanyin Biotech, Shanghai) and overexpression plasmids for c-FOS and p-Smad3 (Jikai Gene, Shanghai) were transfected using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: shRNA, Staining, Immunohistochemical staining, Expressing, TUNEL Assay, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FOS drives podocyte injury and renal fibrosis in diabetic kidney disease via direct Smad3 binding

doi: 10.1007/s00018-026-06215-z

Figure Lengend Snippet: HG promotes EMT and fibrosis progression in HPC and MPC5 cells. ( A , E ) Cell viability of HPC and MPC5 cells treated with different glucose concentrations for 24h, 48h, and 72h. ( B , C , F , G ) Representative flow cytometry plots of apoptosis measured by Annexin V-FITC/PI staining. ( D , H ) Quantification of total apoptotic rates (early+late apoptosis). Data are presented as the mean ±SD from three independent experiments. ( I , K ) Cell migration ability under HG conditions assessed by scratch wound healing and transwell assays. For wound healing, images were captured at 40× magnification with 1 field per replicate (3 biological replicates). For transwell migration, 5 images per replicate were taken at 100× magnification (3 biological replicates). ( J , L ) Western blot showing expression levels of p-FOS, FOS, p-Smad3, Smad3, Podocin, EMT-related markers, and fibrosis-related proteins under HG conditions. HG represents 35mmol/L glucose. **P < 0.01, ***P < 0.001

Article Snippet: Gene-specific siRNAs targeting FOS and Smad3 (Hanyin Biotech, Shanghai) and overexpression plasmids for c-FOS and p-Smad3 (Jikai Gene, Shanghai) were transfected using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Staining, Migration, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FOS drives podocyte injury and renal fibrosis in diabetic kidney disease via direct Smad3 binding

doi: 10.1007/s00018-026-06215-z

Figure Lengend Snippet: FOS-Smad3 interaction drives TGF-β/Smad3-mediated renal EMT and fibrosis. (A–D) Co-IP assays confirming the direct physical interaction between FOS and p-Smad3 in HPC and MPC5 cells under normal control (NC) and HG conditions. (E, F) Smad3 phosphorylation was suppressed upon FOS knockdown. Protein levels of p-Smad3 and total Smad3 were assessed by western blot. (G, H) FOS overexpression upregulated p-Smad3 expression, as shown by western blot. (I, K) Cell migration ability evaluated by scratch wound healing assay and transwell assay across the following groups: HG, OEFOS, Vector, OEFOS + siSmad3, and OEFOS + SIS3. For scratch wound healing assay, images were acquired at 40× magnification with 1 field per replicate (n=3 biological replicates). For transwell assay, images were taken at 100× magnification with 5 fields per replicate (n=3 biological replicates). (J, L) Smad3 knockdown reversed FOS-induced EMT abnormalities and TGF-β/Smad3 pathway activation, as evaluated by western blot. HG represents 35mmol/L glucose. Data are presented as mean ± SD. ***P < 0.001

Article Snippet: Gene-specific siRNAs targeting FOS and Smad3 (Hanyin Biotech, Shanghai) and overexpression plasmids for c-FOS and p-Smad3 (Jikai Gene, Shanghai) were transfected using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Control, Phospho-proteomics, Knockdown, Western Blot, Over Expression, Expressing, Migration, Wound Healing Assay, Transwell Assay, Plasmid Preparation, Activation Assay

Journal: Frontiers in Endocrinology

Article Title: ECM remodeling features in reparative chondrocytes during knee osteoarthritis

doi: 10.3389/fendo.2026.1773139

Figure Lengend Snippet: ECM-related cues promote RepC-associated gene expression and activate TGF-β/SMAD signaling in human articular chondrocytes. (A) Relative mRNA expression levels of RepC-associated genes (CILP, OGN, MMP13, and FRZB) in primary human articular chondrocytes treated with TGF-β1 (5 ng/mL), FN1 (10 μg/mL), or their combination. Gene expression was normalized to internal controls and expressed relative to untreated controls (Con). (B) Quantification of protein expression levels of CILP and MMP13, as well as phosphorylation levels of SMAD2 and SMAD3, under the same treatment conditions as in (A) . Protein levels were normalized to β-actin, and phosphorylation levels were normalized to total SMAD2 or SMAD3, respectively. (C) Representative immunoblot images showing CILP, MMP13, phosphorylated SMAD2 (P-SMAD2), total SMAD2, phosphorylated SMAD3 (P-SMAD3), total SMAD3, and β-actin under control, TGF-β1, FN1, and combined TGF-β1 + FN1 stimulation conditions. Data are presented as mean ± SD from n = 3 independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc multiple-comparison test. **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies included CILP (PA5-18553, Thermo Fisher Scientific; 1:1500), MMP13 (18165-1-AP, Proteintech; 1:1000), phospho-SMAD2 (ET1612-32, HUABIO; 1:5000), phospho-SMAD3 (ET1609-41, HUABIO; 1:5000), SMAD2 (12570-1-AP, Proteintech; 1:2000), SMAD3 (66516-1-Ig, Proteintech; 1:2000), and β-actin (66009-1-Ig, Proteintech; 1:20000).

Techniques: Gene Expression, Expressing, Phospho-proteomics, Western Blot, Control, Comparison