Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Representative immunofluorescence images of primary cilia in healthy control (HC), systemic sclerosis (SSc), and VEDOSS (very early SSc) dermal fibroblasts under basal conditions (CTR) or following 24 h TGFβ stimulation. Acetylated α-tubulin marks the axoneme (red) and nuclei are stained with DAPI (blue). (B) Quantification of primary cilium length in HC, SSc, and VEDOSS fibroblasts under basal conditions. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (C) Time course of TGFβ-induced cilia shortening in HC and SSc fibroblasts. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (D) Quantification of cilium length in HC and SSc fibroblasts subjected to a 24 h TGFβ pulse followed by washout and recovery in 0.5% serum for 24 and 48 h. n=3 experimental repeats. (E) Quantification of cilium length in HC and SSc fibroblasts after sequential serum starvation, TGFβ exposure, and two passages in 10% serum. n=3 experimental repeats. (F) Cilium length in HC and SSc fibroblasts treated with the TGFβ receptor inhibitor SD208 in the presence or absence of exogenous TGFβ. n=3 experimental repeats. (G) Representative western blot of pSMAD3, total SMAD3, and βactin in HC and SSc fibroblasts treated with TGFβ and/or SD208. n=3 donors per group. (H) Quantification of cilium length in HC and SSc fibroblasts cultured in full growth medium in the presence of DMSO (CTR) or SD208 for 2 passages. n=3 experimental repeats. All data panels were analysed by ANOVA (ns, non-significant; ** P<0.01; *** P<0.001; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Immunofluorescence, Control, Staining, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Top: representative western blot showing the effect of SMAD3 siRNA (siSMAD3) on total SMAD3 protein level and on TGFβ-induced SMAD3 phosphorylation in healthy control (HC) and systemic sclerosis (SSc) fibroblasts, compared to scrambled siRNA (siSCR). Bottom: Densitometric quantification of SMAD3 in control (siSCR) and siSMAD3-treated HC and SSc fibroblasts (n=3 individual donors per group). (B) Quantification of cilium length in HC and SSc fibroblasts treated with siSCR or siSMAD3 and stimulated for 24 h with TGFβ. n=3 individual donors per group. (C) Top: representative western blots showing pMLC2, ppMLC2, and total MLC2, in response to treatments TGFβ with or without KD025 in three HC donor and three SSc donor fibroblast lines. Bottom: Densitometric quantification of pMLC2 normalised to MLC2 in the same condition. (D) Cilium length in HC and SSc fibroblasts treated with the ROCK2 inhibitor KD205 ± TGFβ. Mean cilia length was measured in three HC donor and three SSc donor fibroblast lines treated as indicated. All data panels were analysed by ANOVA (ns, non-significant; * P<0.05; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 1. Analysis of Smad3/4 complex binding to the β3GalT4 promoter. (A) Smad4 binding site (5'‑GTCTAGAC‑3') located between positions ‑788 and ‑795, relative to the transcriptional start point of the β3GalT4 promoter. (B) EMSA. Each lane contained 0.3 nM labeled probe. Smad4 protein (900 ng) was loaded. Smad3 (400, 600, 800 and 1000 ng) was loaded on lanes 3‑6, respectively. (B‑D) Black arrow indicates retarded DNA fragments; white arrow indicates free probes. (C) EMSA to eliminate the nonspecific binding of the Smad3/4 proteins. The labeled probe and 100 or 200‑fold excess of the unlabeled probe were used in competitive assays. A fragment of actin promoter was used as a nonspecific probe. (D) Determination of the binding sites of Smad3/4. Top: Nucleotide sequence of part of the β3GalT4 promoter region and SBE. All probes used were 40 bp. NotI sites were generated at the SBE site to produce a mutated probe. Underlined nucleotides were changed: Bottom: EMSA using the unlabeled probe and mutated or intact DNA fragment. (E) ChIP assays. Input DNA, a DNA fragment immunoprecipitated with anti‑Smad4 antibody (lane ‘S’) and a DNA fragment immunoprecipitated with IgG (lanes ‘‑’) were used as templates for the polymerase chain reaction. A nonspecific DNA region was used as a negative control. Anti‑Pol II antibodies that immunoprecipitated the Pol II‑actin promoter complexes were used as a positive control. β3GalT4, β1,3-galactosyltransferase‑4; EMSA, electrophoretic mobility shift assay; SBE, Smad4 binding element.

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Binding Assay, Labeling, Sequencing, Generated, Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Positive Control, Electrophoretic Mobility Shift Assay

Journal: Molecular medicine reports

Article Title: Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

doi: 10.3892/mmr.2014.2912

Figure Lengend Snippet: Figure 2. Effect of Smad3 or Smad4 overexpression in NMuMG cells. (A) Morphological changes. Cells were cultured in 6‑well plates and treated with 2 ng/ ml TGFβ for 48 h. Images were captured under phase‑contrast microscopy. (B and C) Expression of EMT markers in the transfected cells. Cells were cultured in 6‑well plates and treated with or without 2 ng/ml TGFβ for 48 h. The cells were harvested and lysed in radioimmunoprecipitation assay buffer. Lysates (10 µg protein/well) were subjected to SDS‑PAGE and western blot analysis and the expression of the markers N‑cad, vimentin, E‑cad and β‑catenin was analyzed by western blot analysis, as described in Materials and methods. Representative western blot analysis results and expression levels relative to Tubulin are expressed as the mean ± standard deviation from triplicate experiments. *0.01

Article Snippet: The primary antibodies used were mouse anti-E-cad IgG2a monoclonal antibody and mouse-anti-β catenin IgG1 monoclonal antibody (BD Biosciences, San Jose, CA, USA), mouse anti-N-cad IgG1 and rabbit anti-RNA polymerase II (Pol II) IgG polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-vimentin IgG1 monoclonal antibody and anti-β tubulin IgG1 monoclonal antibody (Sigma-Aldrich), rabbit anti-Smad3 mAb IgG monoclonal antibody and anti-Smad4 polyclonal antibody (Cell Signaling Technology, Boston, MA, USA).

Techniques: Over Expression, Cell Culture, Microscopy, Expressing, Transfection, Radio Immunoprecipitation, Western Blot, Standard Deviation