Journal: Cell reports

Article Title: Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule

doi: 10.1016/j.celrep.2019.11.019

Figure Lengend Snippet:

Article Snippet: Rabbit p-SMAD2/3 , R and D systems , Cat# MAB8935; RRID:AB_2313773.

Techniques: Virus, Recombinant, Luciferase, Imaging, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Software

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 2. The expression and the phosphorylation status of Smad2 and Smad3 in the parental, the mock- and ß1A-integrin-transfected HepG2 cells. The cells without TGF-ß1 stimulation were analyzed by Western blotting with anti-Smad2 and anti-Smad3 antibodies. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Expressing, Phospho-proteomics, Transfection, Western Blot

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 3. The modulation of the TGF-ß1-induced phosphorylation of the Smad proteins in the parental, the mock- and ß1A-integrin-transfected HepG2 cells. The cells were treated with 2 ng/ml of TGF-ß1, were harvested at the indicated times and were analyzed by Western blotting using each antibody. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 4. The altered nuclear translocation of the phosphorylated Smad proteins after TGF-ß1 treatment in ß1A-integrin-transfected HepG2 cells. The cells were separated into the cytoplasmic (Cy) and nuclear (Nu) fractions with or without 2 ng/ml of TGF-ß1 for 1 h and were analyzed by Western blotting. Histone H1 was detected as the control for the nuclear proteins. pSmad2C, Smad2 phosphorylated at C-terminal site; pSmad2L, Smad2 phosphorylated at linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Translocation Assay, Transfection, Western Blot, Control

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 5. The involvement of the MAP kinase pathway in the alteration of the Smad phosphorylation in the ß1A-integrin-transfected cells. The HepG2 cells overexpressing ß1A-integrin were treated with PD98509 (ERK inhibitor; left), SB203580 (p38 inhibitor; center) and SP600125 (JNK inhibitor; right) for 6 h prior to the 1-h treatment with 2 ng/ml of TGF-ß1. pSmad2C, Smad2 phosphorylated at the C-terminal site; pSmad2L, Smad2 phosphorylated at the linker region; tSmad2, total Smad2; pSmad3C, Smad3 phosphorylated at the C-terminal site; pSmad3L, Smad3 phosphorylated at the linker region; tSmad3, total Smad3.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Phospho-proteomics, Transfection

Journal: International journal of oncology

Article Title: Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

doi: 10.3892/ijo_00000463

Figure Lengend Snippet: Figure 6. The restoration of the TGF-ß1-induced Smad-mediated transcription by mutant Smads lacking phosphorylation sites in the linker region. The HepG2 cells were transfected with the SBE4 reporter plasmid and the ß1- integrin expression plasmid together with mutant Smads lacking phosphory- lation sites in the linker region (Smad2-EPSM and Smad3-EPSM) were then treated with 2 ng/ml of TGF-ß1 for 24 h. The data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 compared with control without Smad2-EPSM and Smad3-EPSM.

Article Snippet: The antibodies against the phosphorylated form of the C-terminal region of Smad2 (Ser465/467) and Smad3 (Ser423/425), as well as the antibody against the phosphorylated form of the linker region of Smad2 (Ser245/250/255), were purchased from Cell Signaling (Boston, MA).

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing, Control

Journal: Science Advances

Article Title: Divergent stem cell mechanisms govern the primary body axis and appendage regeneration in the axolotl

doi: 10.1126/sciadv.adx5697

Figure Lengend Snippet: ( A ) UMAP visualization of refined subclusters within the regeneration active zone. MuSC, IM1, and IM2 are partitioned into nine subclusters based on shared gene expression programs between progenitors and their progeny. Each cell type, e.g., IM2, is subdivided into subclusters committed to myoblasts (IM2_Myo), fibroblast 2 (IM2_Fib2), or uncommitted (IM2_UC). ( B ) Heatmap showing the scores of predicted pathway activity in nine defined subclusters from (A), combined with Fib1, Fib2, and Myo. ( C and D ) SB-431542 inhibits the production of fibroblasts during tail regeneration. Images of CHERRY fluorescence of 26-day tail regenerates treated with SB-431542 (D) and control (C) ( n = 3 each). ( E and F ) Quantification of CHERRY signal area in the fin and regenerated fin length following SB-431542 treatment ( n = 3). dpt, days posttreatment. Error bars, SEM; statistical analysis was conducted using Student’s t test (E) and one-way analysis of variance (ANOVA) (F); ** P < 0.01, **** P < 0.0001; ns, not significant. ( G ) p-SMAD2 and SMAD2 immunoblots of regenerated tails treated with SB-431542 and control ( n = 3 each). Glyceraldehyde-3-phosphate dehydrogenase immunoblot, loading control. ( H and I ) Quantification of signal intensity from p-SMAD2 and SMAD2 immunoblots in (G) ( n = 3 each). Student’s t test; ** P < 0.01. ( J ) Boxplot of the scores of endogenous TGF-β activity in MuSCs. Note that scores are attenuated in IM2 committed to myoblasts (IM2_Myo), in contrast, elevated in IMs to fibroblasts (IM1_Fib1, IM2_Fib2), compared to that in MuSCs at 0 days postamputation (dpa). ( K and L ) Cell fate trajectory predictions following in silico perturbation of TGF-β signaling. Activation of TGF-β diverts cells from muscle-related lineages to fibroblast-related lineages (K), whereas its suppression diverts cells from fibroblast-related lineages to muscle-related lineages (L). Rectangles in (C) and (D) are shown at higher magnification. Scale bars, 1 mm [(C) and (D)]. See also fig. S20.

Article Snippet: The primary antibodies used in this study included mouse–anti-TUJ1 (R&D, MAB1195), rabbit–anti-CD34 (Abcam, ab81289), rabbit–anti-MEF2C (Santa Cruz Biotechnology, sc-313), rabbit–anti-SOX2 (GeneTex, GTX124477), rabbit–anti-PRRX1 (a gift from E. M. Tanaka’s laboratory), rabbit–anti-SOX10 (Abcam, ab264405), mouse–anti-PAX7 (DSHB), rat–anti-CHERRY (Invitrogen, M11217 ), rabbit–anti-SOX9 (Merck, AB5535), rabbit–anti-Laminin (Sigma-Aldrich, L9393), mouse–anti-MHC [fluorescein isothiocyanate (FITC)–coupled, DSHB], and rabbit–anti–p-SMAD2 (CST, 18338 T).

Techniques: Gene Expression, Activity Assay, Fluorescence, Control, Western Blot, In Silico, Activation Assay

Journal: Journal of Translational Medicine

Article Title: Integrative transcriptomic and experimental analysis identifies GREM1 as a pro-metastatic mediator in lung adenocarcinoma

doi: 10.1186/s12967-026-08793-9

Figure Lengend Snippet: GREM1 interacts with BMP2 and regulates TGF-β/SMAD signaling pathway. ( A ) schematic diagram illustrates protein-protein interaction networks involving hub genes and EMT, glycolysis and TGF-β signaling pathways genes, based on findings from the STRING database. ( B ) 3D Binding model analysis(GREM1 in pink and BMP2 in green). ( C ) Immunofluorescence double staining detecting the colocalization and expression changes of GREM1 (red) and BMP2 (green) in the control group, GREM1 knockdown group (sh-GREM1-1), and overexpression group (oe-GREM1). ( D ) Co-immunoprecipitation assay verifying the direct binding between GREM1 and BMP2. ( E ) Western blot analysis of TGF-β/SMAD pathway components, including SMAD2/3, p-SMAD2, p-SMAD3, TGF-β1, BMP2, Smad1, Smad5 and Smad8. ( F ) IHC staining of GREM1 in tumor and adjacent normal tissues. ( G ) Representative images of GREM1 expression in immunohistochemical staining. ( H ) Kaplan–Meier curves for overall survival by GREM1 expression

Article Snippet: The protein was separated, transferred, and then incubated with 5% milk for 2 h. Antibodies including GREM1 (Santa Cruz, sc-515877), TGF-β1 (Santa Cruz, sc-130348), smad23 (Santa Cruz, sc-398844), p-smad2 (ABclonal, AP1007), p-smad3 (ABclonal, AP0727), smad1 (ABclonal, A19113), smad5 (ABclonal, A22749), smad8 (Santa Cruz, sc-518051), BMP2(ABclonal, A27101 ), snail(ABclonal, A11794), E-cadherin (Proteintech, Cat No. 20874-1-AP), N-cadherin (proteintech, Cat No. 22018-1-AP), Vimentin (Santa Cruz, sc-373717), HK2 (Proteintech, Cat No. 22029-1-AP), LDHA (ABclonal, A21893), PKM2 (ABclonal, A0268), TPI1(ABclonal, A2579) and β-Actin (ABclonal, AC038) were incubated with the protein overnight at 4 °C.

Techniques: Protein-Protein interactions, Binding Assay, Immunofluorescence, Double Staining, Expressing, Control, Knockdown, Over Expression, Co-Immunoprecipitation Assay, Western Blot, Immunohistochemistry, Immunohistochemical staining, Staining