bio techne r d system  (R&D Systems)

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: Chol-Loading Downregulates TGFβ Signaling in hVSMC hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT; ie, 0 μg/mL cholesterol) for 24 hours in the presence or absence of TGFβ1 ligand (10 pg/mL). Total RNA was isolated and quantitative polymerase chain reaction (qPCR) was performed to determine the pri-Mir143/145 precursor transcripts (A,B) or SMC markers, Acta2 and Tagln (C,D). hVSMCs were treated as in A and B, but either in the presence or absence of TGFβ1 10 pg/mL) and/or nonscrambled (NS) or Mir145 mimic (60 nmol/L). qPCR was performed to determine expression of Acta2 (E) and (F) Srf mRNA. (G) hVSMCs were treated as in A and B, but either in the presence or in absence of TGFβ1 (10 pg/mL) and/or Mir145 inhibitor (60 nmol/L). qPCR was performed to determine expression of Acta2. (H) Immunofluorescence images of total SMAD2/3 (green) in hVSMCs after 24 hours of the indicated treatments. Cytoplasm was stained with phalloidin (red). Nuclei were determined as phalloidin negative area (bar = 50 μm). (I) hVSMCs were treated as in A and B, but with varying amounts of Chol and in the presence or absence of recombinant TGFβ1 (10 pg/mL) for 24 hours. Proteins were extracted for Western blotting to detect phosphorylated (p) SMAD2/3, and α-SMA. Total SMAD2/3 or GAPDH was used as loading CT proteins. Blots are representative of at least 3 independent experiments, and the replicates were quantified by densitometry. For data comparisons of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Data are presented as the mean ± SEM of 3 independent experiments, and P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). ns = not significant; other abbreviations as in .

Article Snippet: The primary antibodies used were as follows: ACTA2 (#A2547, Sigma); CNN1 (#M3556, DAKO); SRF (#5147, Cell Signaling); p38MAPK (#sc-535, Santa Cruz Biotechnology); SMAD2/3 (#8685, Cell Signaling); TUBA (#T-5168, Sigma); and SMAD2 (#3103, Cell Signaling), phospho-SMAD2 (#3101S, Cell Signaling), phospho-p38MAPK (#9211S, Cell Signaling), SMAD4 (#9515, Cell Signaling); CD68 (#MCA1815, AbD Serotec, Bio-Rad); KLF4 (#12173, Cell Signaling); PU.1 (#sc-352, Santa Cruz Biotechnology); TGFβR1 (#3712, Cell Signaling); TGFβR2 (#sc-400, Santa Cruz Biotechnology); Caveolin (#610059, BD Transduction Laboratories); CD71 (#13113, Cell Signaling); GAPDH (#AM4300, Ambion).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Recombinant, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: HDL Treatment In Vitro Restores TGFβ Signaling in Cholesterol-Loaded hVSMCs (A) hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin for 24 hours, followed by high-density lipoprotein (HDL) (50 μg/mL) treatment for 48 hours. Then, treatment groups were stimulated with recombinant TGFβ1 (10 pg/mL). Western blotting was performed to detect pSMAD2 and total (t) SMAD2, with densitometry used for quantification. (B to E) qPCR was performed to detect expression of Mir143/145, Myocd, Acta2, Cnn1, and Hmgcr at the conclusion of the experiment in A. (F) Chol-loaded cells were either treated with HDL alone, HDL + TGFβR1 antagonist (TGFβR1i; 50 ng/mL), or left untreated. Western blotting was performed to detect α-SMA. GAPDH was used as loading CT protein. For data analysis of 2 or more independent groups, 1-way or 2-way analysis of variance followed by Dunnett post hoc test was performed. Blots are representative of 3-5 independent experiments (mean ± SEM). P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). Abbreviations as in and .

Article Snippet: The primary antibodies used were as follows: ACTA2 (#A2547, Sigma); CNN1 (#M3556, DAKO); SRF (#5147, Cell Signaling); p38MAPK (#sc-535, Santa Cruz Biotechnology); SMAD2/3 (#8685, Cell Signaling); TUBA (#T-5168, Sigma); and SMAD2 (#3103, Cell Signaling), phospho-SMAD2 (#3101S, Cell Signaling), phospho-p38MAPK (#9211S, Cell Signaling), SMAD4 (#9515, Cell Signaling); CD68 (#MCA1815, AbD Serotec, Bio-Rad); KLF4 (#12173, Cell Signaling); PU.1 (#sc-352, Santa Cruz Biotechnology); TGFβR1 (#3712, Cell Signaling); TGFβR2 (#sc-400, Santa Cruz Biotechnology); Caveolin (#610059, BD Transduction Laboratories); CD71 (#13113, Cell Signaling); GAPDH (#AM4300, Ambion).

Techniques: In Vitro, Recombinant, Western Blot, Expressing

Journal: JACC: Basic to Translational Science

Article Title: HDL Regulates TGFβ-Receptor Lipid Raft Partitioning, Restoring Contractile Features of Cholesterol-Loaded Vascular Smooth Muscle Cells

doi: 10.1016/j.jacbts.2025.101461

Figure Lengend Snippet: HDL Treatment Displaces TGFβ Receptor From Membrane Lipid Rafts in Chol-Loaded hVSMCs and Restores its Signaling hVSMCs were treated with Chol (5 μg/mL) or 0.2% bovine serum albumin (CT) for 24 hours, after which they were all treated with HDL (50 μg/mL) for 24 hours. (A) At the end of the 48-hour protocol, LR and NR fractions were isolated, and Western blotting was performed using each of these fractions to determine the expressions of TGFβR1 and TGFβR2, as well as CAV1, and flotillin. Densitometry was performed to quantify the level of (B) TGFβR1 and (C) TGFβR2. (D) hVSMCs were loaded with Chol (48 hours, 5 μg/mL) and were then either treated with HDL (50 μg/mL) for 24 hours, or left untreated. Western blotting was performed to determine pSMAD2, SMAD2, and GAPDH levels. For data analysis of 2 or more independent groups, 2-way analysis of variance followed by Šídák multiple comparisons post hoc test was performed. Data are presented as the mean ± SEM of at least 3 independent experiments, and the P values are as indicated (∗ P < 0.05, ∗∗ P < 0.01). Abbreviations as in , , , .

Article Snippet: The primary antibodies used were as follows: ACTA2 (#A2547, Sigma); CNN1 (#M3556, DAKO); SRF (#5147, Cell Signaling); p38MAPK (#sc-535, Santa Cruz Biotechnology); SMAD2/3 (#8685, Cell Signaling); TUBA (#T-5168, Sigma); and SMAD2 (#3103, Cell Signaling), phospho-SMAD2 (#3101S, Cell Signaling), phospho-p38MAPK (#9211S, Cell Signaling), SMAD4 (#9515, Cell Signaling); CD68 (#MCA1815, AbD Serotec, Bio-Rad); KLF4 (#12173, Cell Signaling); PU.1 (#sc-352, Santa Cruz Biotechnology); TGFβR1 (#3712, Cell Signaling); TGFβR2 (#sc-400, Santa Cruz Biotechnology); Caveolin (#610059, BD Transduction Laboratories); CD71 (#13113, Cell Signaling); GAPDH (#AM4300, Ambion).

Techniques: Membrane, Isolation, Western Blot