tgf βr ii  (Bioss)

Journal: bioRxiv

Article Title: Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1

doi: 10.1101/2025.02.21.639264

Figure Lengend Snippet: (A and B) Secreted TGF-β1 quantification by ELISA assay on supernatants from D0 to D7 HuMoSC (A) and supernatant from MCF7 cells alone or in co-culture with HuMoSC, with or without treatment with Ly2109761 (B). Secretion was normalized to D0-monocyte secretion (A). “MCF7 medium” condition correspond to basal TGF-β detected in the medium (B). (C) Heatmap comparing all significant surfaceome changes in HuMoSC compared to monocyte. (D) TGF-β western blot using Monocyte or HuMoSC total protein fractions before streptavidin pulldown (Whole Cell Lysate) or cell surface protein fractions of biotinylated proteins only (Cell Surface Fraction). α-Tubuline was used as a control for whole cell lysate loading. (E) Tumorsphere formation assay of MCF7 cells alone or after co-culture with CD52 + HuMoSC and treated or not with 5 μM TGF-βRI/II inhibitor Ly2109761 or 1 μg/mL αTGF-β neutralizing antibody. (F) Tumorsphere formation assay of MCF7 cells alone after in co-culture with CD33 high CD52 + myeloid cells isolated from pleural effusion of breast cancer patients and differentially expressing CD33 (high or low) and CD52 and treated or not with 5 μM TGF-βRI/II inhibitor Ly2109761 or 1 μg/mL αTGF-β neutralizing antibody. Data are means ± S.E.M. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, not significant. Kruskal-Wallis test with Dunn’s multiple comparisons post-test (A-B, E-F). Data are means of n = 3 independent biological replicates. (C) Log2 FC (HuMoSC/Monocyte) above 2 or below -2 (4-fold) were considered as significantly upregulated (red) or downregulated (blue), respectively. P-values were determined by unpaired two-tailed Student’s t-tests; P < 0.05.

Article Snippet: When indicated, monocytes, HuMoSC or patient’s myeloid cells were added in each well at a 1:5 ratio (1.500 cells per well) and wells were treated with 5 μM TGF-βR type I/II inhibitor Ly2109761 (MedChemExpress) or TGF-β1,2,3 antibody (MAB1835, R&D Systems) at neutralizing concentration (1 μg/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Western Blot, Control, Tube Formation Assay, Isolation, Expressing, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of miR-155-5p Exerts Anti-Fibrotic Effects in Silicotic Mice by Regulating Meprin α

doi: 10.1016/j.omtn.2019.11.018

Figure Lengend Snippet: Recombinant Meprin α Inhibits Activation of NR8383 Macrophages Induced by SiO 2 (A) Protein expression of meprin α, MCP-1, TGF-β1, TGF-βR I, TGF-βR II, and p-Smad2/3 was measured in NR8383 cells treated with 10, 25, and 50 μg/cm 2 silica. * Compared with control group, p < 0.05. (B) Expression of pro-COL I, MCP-1, TGF-β1, TGF-βR I, TGF-βR II, and p-Smad2/3 was measured in NR8383 cells treated with SiO 2 , SiO 2 plus meprin α, and SiO 2 plus actinonin. * Compared with control group, p < 0.05; # Compared with SiO 2 group, p < 0.05. Data are presented as the mean ± SD. n = 3 per group.

Article Snippet: Protein samples (10 μg) were solubilized in 5× sample buffer (AS0001-5, Seracare, USA), heated at 95°C for 10 min, centrifuged at 3,000 × g for 1 min, loaded on a 12% Tris-HCl-SDS-polyacrylamide gel, and separated for 1 h at 120 V. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (31337600, Roche Diagnostics, Germany) and then blocked with 5% BSA for 1 h at room temperature, followed by incubation overnight at 4°C with a specific primary antibody against meprin α, COL I (ab34710, Abcam), α-SMA, MCP-1 (DF7577, Affinity, USA), TGF-β1 (ARG56894, Arigo Biolaboratories, Taiwan, China), TGF-βR I (A16983, ABclonal, Wuhan, China), TGF-βR II (ARG59501, Arigo), p-Smad2/3 (8828s, Cell Signaling Technology, MA, USA), or Smad2/3 (5678, Cell Signaling Technology), followed by incubation with goat anti-rabbit or anti-mouse secondary antibodies (074-1506/074-1806, Kirkegaard & Perry Laboratories, USA) at a dilution of 1:5,000 in blocking buffer.

Techniques: Recombinant, Activation Assay, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of miR-155-5p Exerts Anti-Fibrotic Effects in Silicotic Mice by Regulating Meprin α

doi: 10.1016/j.omtn.2019.11.018

Figure Lengend Snippet: Recombinant Meprin α Inhibits the Fibrotic Response in Fibroblasts Treated with CM of Silica-Treated Macrophages (A) Protein expression of pro-COL I, α-SMA, TGF-βR I, TGF-βR II, and p-Smad2/3 was measured in lung fibroblasts treated with CM plus meprin α or actinonin. Data are presented as the mean ± SD. n = 3 per group. * Compared with control group, p < 0.05; # Compared with CM group, p < 0.05. (B) Expression of α-SMA in lung fibroblasts observed by IHC staining (Bar = 100 μm).

Article Snippet: Protein samples (10 μg) were solubilized in 5× sample buffer (AS0001-5, Seracare, USA), heated at 95°C for 10 min, centrifuged at 3,000 × g for 1 min, loaded on a 12% Tris-HCl-SDS-polyacrylamide gel, and separated for 1 h at 120 V. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (31337600, Roche Diagnostics, Germany) and then blocked with 5% BSA for 1 h at room temperature, followed by incubation overnight at 4°C with a specific primary antibody against meprin α, COL I (ab34710, Abcam), α-SMA, MCP-1 (DF7577, Affinity, USA), TGF-β1 (ARG56894, Arigo Biolaboratories, Taiwan, China), TGF-βR I (A16983, ABclonal, Wuhan, China), TGF-βR II (ARG59501, Arigo), p-Smad2/3 (8828s, Cell Signaling Technology, MA, USA), or Smad2/3 (5678, Cell Signaling Technology), followed by incubation with goat anti-rabbit or anti-mouse secondary antibodies (074-1506/074-1806, Kirkegaard & Perry Laboratories, USA) at a dilution of 1:5,000 in blocking buffer.

Techniques: Recombinant, Expressing, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of miR-155-5p Exerts Anti-Fibrotic Effects in Silicotic Mice by Regulating Meprin α

doi: 10.1016/j.omtn.2019.11.018

Figure Lengend Snippet: miR-155-5p Decreases the Levels of Meprin α and Promotes Macrophage Activation Induced by SiO 2 Levels of meprin α, MCP-1, TGF-β1, TGF-βR I, TGF-βR II, and p-Smad2/3 were measured in NR8383 cells treated with SiO 2 , and treated with agomiR-155-5p or antamiR-155-5p. Data are presented as the mean ± SD. n = 3 per group.

Article Snippet: Protein samples (10 μg) were solubilized in 5× sample buffer (AS0001-5, Seracare, USA), heated at 95°C for 10 min, centrifuged at 3,000 × g for 1 min, loaded on a 12% Tris-HCl-SDS-polyacrylamide gel, and separated for 1 h at 120 V. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (31337600, Roche Diagnostics, Germany) and then blocked with 5% BSA for 1 h at room temperature, followed by incubation overnight at 4°C with a specific primary antibody against meprin α, COL I (ab34710, Abcam), α-SMA, MCP-1 (DF7577, Affinity, USA), TGF-β1 (ARG56894, Arigo Biolaboratories, Taiwan, China), TGF-βR I (A16983, ABclonal, Wuhan, China), TGF-βR II (ARG59501, Arigo), p-Smad2/3 (8828s, Cell Signaling Technology, MA, USA), or Smad2/3 (5678, Cell Signaling Technology), followed by incubation with goat anti-rabbit or anti-mouse secondary antibodies (074-1506/074-1806, Kirkegaard & Perry Laboratories, USA) at a dilution of 1:5,000 in blocking buffer.

Techniques: Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of miR-155-5p Exerts Anti-Fibrotic Effects in Silicotic Mice by Regulating Meprin α

doi: 10.1016/j.omtn.2019.11.018

Figure Lengend Snippet: miR-155-5p Decreases the Levels of Meprin α and Promotes Fibroblast Activation Induced by CM of Macrophages (A) Levels of meprin α, MCP-1, TGF-β1, TGF-βR I, TGF-βR II, and p-Smad2/3 were measured in fibroblasts treated with CM derived from silica-treated NR8383 cells, and treated with agomiR-155-5p or antamiR-155-5p. Data are presented as the mean ± SD. n = 3 per group. (B) Expression of α-SMA in lung fibroblasts observed by IHC staining (scale bars, 100 μm).

Article Snippet: Protein samples (10 μg) were solubilized in 5× sample buffer (AS0001-5, Seracare, USA), heated at 95°C for 10 min, centrifuged at 3,000 × g for 1 min, loaded on a 12% Tris-HCl-SDS-polyacrylamide gel, and separated for 1 h at 120 V. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (31337600, Roche Diagnostics, Germany) and then blocked with 5% BSA for 1 h at room temperature, followed by incubation overnight at 4°C with a specific primary antibody against meprin α, COL I (ab34710, Abcam), α-SMA, MCP-1 (DF7577, Affinity, USA), TGF-β1 (ARG56894, Arigo Biolaboratories, Taiwan, China), TGF-βR I (A16983, ABclonal, Wuhan, China), TGF-βR II (ARG59501, Arigo), p-Smad2/3 (8828s, Cell Signaling Technology, MA, USA), or Smad2/3 (5678, Cell Signaling Technology), followed by incubation with goat anti-rabbit or anti-mouse secondary antibodies (074-1506/074-1806, Kirkegaard & Perry Laboratories, USA) at a dilution of 1:5,000 in blocking buffer.

Techniques: Activation Assay, Derivative Assay, Expressing, Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-βR II and NKG2D

doi: 10.1007/s00262-017-1959-1

Figure Lengend Snippet: Generation of NK-92 cells expressing chimeric TN antigen receptors. a Schematic representation of the TN chimeric receptor. TN chimeric receptor includes extracellular and transmembrane domains of TGF-βR II and intracellular domain of NKG2D. b Expression of YFP and CD56 in NK92, NK-92-TN, and NK-92-Vector cells analyzed by flow cytometry using PE-labeled anti-CD56 antibody. c Expression of YFP and surface TGF-βR in NK92, NK-92-TN, and NK-92-Vector cells analyzed by flow cytometry using PE-labeled anti-TGF-βR II antibody. The mean fluorescence of TGF- βR II expression was also shown. Data shown are the representatives of at least three independent experiments

Article Snippet: Single-cell suspensions of NK-92, NK-92-TN, or control NK-92-Vector cells were incubated for 30 min at 4 °C with monoclonal antibody (mAb) against TGF-βR II (Abcam, Cambridge, MA).

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Labeling, Fluorescence