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: Oncogene

Article Title: Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling

doi: 10.1038/onc.2017.33

Figure Lengend Snippet: OSM-induced acquisition of CSC properties requires SMAD3. CD24+/CD44− epithelial/non-CSC expressing short hairpin RNA (shRNA) targeting SMAD2 (shSMAD2), SMAD3 (shSMAD3), SMAD4 (shSMAD4) or green fluorescent protein (shGFP) as a control were analyzed with control DNSTAT3 cells by ( a ) western blot analysis to confirm knockdown, or ( b ) flow cytometry before and after OSM exposure for 2 weeks. ( c ) CD24+/CD44− epithelial/non-CSCs were exposed to OSM or TGF-β for 3 weeks and analyzed by microarray hybridization. Gene expression was analyzed using GenomeStudio from Illumina identifying genes that changed in expression from untreated cells at least fivefold. The representation factor and P -value for overlapping expression were calculated using a Fisher’s exact test. ( d ) Transformed HMECs were plated in the presence and absence of either OSM or TGF-β1 for 0.5, 1 and 2 h. Western analysis (top) of whole-cell lysates assessed the proteins levels of Snail, phosphorylated-STAT3 Y705 (P-STAT3), STAT3, phosphorylated SMAD3 S423/425 (P-SMAD3), SMAD3 and Actin (as a loading control). Single-gene qRT-PCR analysis (bottom) using mRNA harvested from cells and primers targeting each specific gene assessed the fold-change expression levels of SNAI1 , HAS2 , SOCS3 and SERPINE1 . ( e ) CD24+/CD44− cells were treated with OSM or TGF-β for 2 weeks, and western blot analysis for phosphorylated SMAD3 S423/425 (P-SMAD3), phosphorylated-SMAD2 S465/467 (P-SMAD2) and Actin (as a loading control) was performed.

Article Snippet: Retroviruses were generated and cells were transduced as previously described., , , , Retroviral constructs encoding DNSTAT3, DNTGFβRII and SMAD7 are described previously., The short hairpins pRetroSUPER targeting SMAD2 (15725), SMAD3 (15726) and SMAD4 (15727) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, shRNA, Control, Western Blot, Knockdown, Flow Cytometry, Microarray, Hybridization, Gene Expression, Transformation Assay, Quantitative RT-PCR

Journal: Oncogene

Article Title: Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling

doi: 10.1038/onc.2017.33

Figure Lengend Snippet: OSM-induced EMT and acquisition of CSC properties requires SMAD3 transcriptional activity in the nucleus. ( a ) CD24+/CD44− cells were exposed to OSM alone or in combination with RUX (10 μM) or SB431542 (SB; 10 μ M ) for 2 h, and then protein levels and gene expression were assessed by western blot analysis (top) and qRT-PCR analysis (bottom), respectively. ( b ) CD24+/CD44− cells were treated with SB431542 (SB) or TGF-β for 2 h and subjected to nuclear protein fractionation, and then western blot analysis of nuclear lysates for P-SMAD3 and Lamin B2 (as a loading control) was performed. ( c ) CD24+/CD44− epithelial/non-CSC expressing shGFP, shSMAD3, shSMAD4, or DNSTAT3 were exposed to OSM for 2 h, and then western blot analysis (top) assessed P-STAT3, STAT3, and Actin (as a loading control) protein levels; and qRT-PCR analysis (bottom) assessed SNAI1 and SOCS3 gene expression. ( d ) Chromatin immunoprecipitation (ChIP) and qRT-PCR analysis for SMAD3 enrichment on the SNAI1 and HAS2 gene promoters in CD24+/CD44− epithelial/non-CSC exposed to SB431542 for 24 h, and then treated with OSM alone, SB431542 alone or in combination for 1 h. ( e ) CD24+/CD44− epithelial/non-CSCs were exposed to OSM or TGF-β for 30 min, and then nuclear localization of SMAD3 (left) and STAT3 (right) was assessed by confocal microscopy (left panel) and quantified (right panel). Red triangles: cells with elevated nuclear localization of both SMAD3 and STAT3. Blue triangles: cells with low levels of SMAD3 and STAT3 nuclear localization ( n =53 for the untreated and TGFβ-treated cells and n =74 for OSM-treated cells). ( f , g ) HPAC cells expressing SMAD7 or empty control Vector were treated with OSM for 7 days, and then ( f ) CD24/44 surface expression was determined via flow cytometry; in addition ( g ) protein levels of phosphorylated-STAT3 Y705 (P-STAT3), Snail and Actin (as a loading control) were determined by western blot analysis. ( h ) HPAC cells expressing shRNA targeting SMAD2 (S2), SMAD3 (S3) or GFP as a control were exposed to OSM for 7 days. Single-gene qRT-PCR analysis (top) assessed SNAI1 gene expression, and western blot analysis (bottom) confirmed SMAD2 and SMAD3 knockdown with Actin used as a loading control.

Article Snippet: Retroviruses were generated and cells were transduced as previously described., , , , Retroviral constructs encoding DNSTAT3, DNTGFβRII and SMAD7 are described previously., The short hairpins pRetroSUPER targeting SMAD2 (15725), SMAD3 (15726) and SMAD4 (15727) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Activity Assay, Gene Expression, Western Blot, Quantitative RT-PCR, Fractionation, Control, Expressing, Chromatin Immunoprecipitation, Confocal Microscopy, Plasmid Preparation, Flow Cytometry, shRNA, Knockdown

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