Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Validation of the differential expression of miR-130a-3p in the livers of mice and patients. (a ) miR-130a-3p expression was decreased, and the expression of TGFBR1, TGFBR2, Col-1, and Col-4 was increased in the liver tissues of the MCD-fed mice. TGFBR1, TGFBR2, Col-1, and Col-4 expression was detected by IHC ( × 200 magnification), and miR-130a-3p expression was detected using an ISH ( × 400 magnification) assay. Positive staining is indicated by a brown color. (b) Effects of the MCD diet on serum ALT and AST levels. Values represent the mean±S.D., *** P <0.001 compared with control. (c) Hepatic mRNA and (d) protein expression of TGFBR1, TGFBR2, Smad2, Smad3, Col-1, and Col-4 were upregulated in the MCD-fed mice compared with the control mice. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control. (e) Histopathological changes were evaluated with hematoxylin and eosin staining, and Masson’s trichrome staining. miR-130a-3p expression was decreased, and TGFBR1, TGFBR2, Col-1, and Col-4 levels were increased in the liver tissues of patients with NASH-related liver fibrosis. The expression of miR-130a-3p was detected by ISH ( × 400 magnification), and TGFBR1, TGFBR2, Col-1, and Col-4 were detected using an IHC ( × 200 magnification) assay. Positive staining is indicated by a brown color

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Biomarker Discovery, Quantitative Proteomics, Expressing, Immunohistochemistry, In Situ Hybridization, Staining, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: miR-130a-3p expression was downregulated and regulated the downstream expression of genes of the TGF-β/SMAD signaling pathway in activated HSCs. ( a ) miR-130a-3p expression was examined by real-time qRT-PCR during HSC activation. Values represent the mean±S.D., (* P <0.05, ** P <0.01). ( b ) The hepatic mRNA and ( c ) protein expression of TGF- β 1 , α -SMA, Smad2, and Smad3 were increased during the process of HSC activation. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( d ) HSC-T6 cells were transfected with miR-130a-3p mimics or mimics control for 48 h. The mRNA and protein ( e ) levels of TGF- β 1 , Smad2, and Smad3 were analyzed by qRT-PCR and western blotting, respectively. Mimics control is the negative control of mimics, and control is the blank control in this group. The result showed that there was no significant difference between control and mimics control. β -actin was used as a loading control. Values represent the mean±S.D., ** P <0.01, *** P <0.001 compared with control. ( f ) The mRNA and protein ( g ) levels of MMP-2, MMP-9, Col-1, and Col-4 were analyzed by qRT-PCR and western blotting, respectively. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Control, Transfection, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Knockdown of either TGFBR1 or TGFBR2 inhibited the TGF- β / SMAD signaling pathway and reduced HSC activation. ( a ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( b ) of TGFBR1 and TGFBR2. β-actin was used as a loading control. Values represent the mean ±SD, *** P < 0.001 compared with control. ( c ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( d ) of α -SMA, Smad2, and Smad3. β-actin was used as a loading control. Values represent the mean ±SD, * P <0.05, ** P <0.01, *** P <0.001 compared with control. ( e ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein levels ( f ) of MMP-2, MMP-9, Col-1, and Col-4. β -actin was used as a loading control. Values represent the mean±S.D., *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Activation Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Overexpression of TGFBR1 and TGFBR2 rescued miR-130a-3p-impaired proliferation and cell growth in HSCs. The HSC-T6 cells were transduced with the miR-130a-3p mimics and the TGFBR1-expressing and TGFBR2-expressing vectors. The rescue efficiency of TGFBR1 or TGFBR2 in the HSC-T6 cells was confirmed by ( a ) RT-PCR and ( b ) western blotting. β -actin was used as a loading control. Values represent the mean±S.D. (*** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the (c) mRNA and ( d ) protein expression of Smad2 and Smad3. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the ( e ) mRNA and ( f ) protein expression of Col-1 and Col-4. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( g ) A schematic diagram highlighting the regulation of HSCs by miR-130a-3p during the genesis and resolution of liver fibrosis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Over Expression, Transduction, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Primers for quantitative real-time PCR analysis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction

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: Developmental cell

Article Title: Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases.

doi: 10.1016/j.devcel.2007.09.001

Figure Lengend Snippet: Figure 4. Dissociation of SEPT2 from Myosin II Alters Myosin II Activity and Increases the Number of Binucleated Cells (A) Representative western blots of CHO-K1 whole-cell lysates prepared after incubating cells with PBS, the TAT-CCT3-pt, or TAT-CCT5 for 1.5 hr. Each lysate was probed with antibodies against the indicated proteins. (B) Quantification of the western blots showing relative levels of myosin II, MYPT1, pMYPT1, SEPT2, SEPT9, MLC, pMLC, and ppMLC. Each exper- iment was done a minimum of three times, normalized to PBS transduction. The error bars reflect the standard deviation. *p < 0.05. (C) Representative western blots of CHO-K1 whole-cell lysates prepared after transfecting with nonspecific shRNAi (shRNAi Scramble) or shRNAi silencing SEPT2. Each lysate was probed with antibodies against the indicated proteins. (D) A representative recording of cell division shown over time depicting cells that were transduced with the TAT-CCT5 or TAT-CCT3-pt. Recordings were started at the beginning of karyokinesis. The number on each panel indicates the time elapsed from the starting point, expressed in minutes.

Article Snippet: Collected samples were probed for SEPT9 (1:1000; Surka et al., 2002), SEPT2 (1:1000), myosin II (1:1000), MYPT1 or pMYPT1 (1:1000; Upstate Biotechnology), and pMLC or ppMLC (1:1000; Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Transduction, Standard Deviation

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