Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 3. Confocal microscopy and cell viability immediately after impact injury. (A) Confocal micrographs show live (green) and dead (red) chondrocytes in an impact site in an un-treated control explant, and in explants treated with 10 mM SFKi and either 10 or 100 mM FAKi. Compared to control, fewer dead chondrocytes were observed in SFKs or FAKi treated groups. (B) Statistical analysis revealed that chondrocyte viability was significantly higher in SFKs or FAKi treated explants compared to control. Between two tested concentrations, 100 mM FAKi was more effective than 10 mM. Asterisk represents statistically significant (p < 0.05, p < 0.01). Bars ¼ 500 mm.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Confocal Microscopy, Control

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 5. Kinetics of FAK and SFKi by western blot analysis. (A) Immunoblot analysis showed that treatment with 10 ng/ml IL-1b and 100 ng/ml TNF-a for 30 min significantly increased SFKs phosphorylation at Tyr 416. 0.1, 1, and 10 mM SFKi diminished this response dose dependently. (B) Analysis of the integrated densities of the bands with the phosphor- to total SFKs ratio. C represents untreated control and Cþ represents 10 ng/ml IL-1b and 100 ng/ml TNF-a treated only. (C) Immunoblots show that treatment with 100 nM fMLF for 30 min did not enhance FAK phosphorylation at Tyr 397; however 100 mM FAKi significantly reduced FAK phosphorylation among tested concentrations of 1, 10, and 100 mM. (D) Analysis of the integrated densities of the bands with the phosphor- to total FAK ratio. C represents untreated control and Cþ represents 100 nM fMLF treated only.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: Logic-based mechanistic machine learning on high-content images reveals how drugs differentially regulate cardiac fibroblasts

doi: 10.1101/2023.03.01.530599

Figure Lengend Snippet: A) 137 single-cell fibroblast features that quantify protein intensity, protein localization, cell morphology, and fiber texture. This heatmap was organized on treatment and feature axes by agglomerative hierarchical clustering. B) Principal component scores of experimental data reduced to a set of 18 representative fibroblast features. C) Principal component loadings the reduced of PCA scores and loadings define a primary axis of fibroblast activation with correlated protein expression of procollagen, αSMA, and F-actin that is modulated by many drugs. Off-axis, the Src inhibitor WH-4-023 modulated the cell texture feature Actin Long Angular Second Moment, which motivated further study.

Article Snippet: The drugs with their respective concentrations are as follows: [0.25,1,2] μg/ml of anakinra (Kineret, SOBI Inc.), [1,5,10] μM valsartan (Sigma-Aldrich, SML0142-10MG), [0.2,1,2] μM BNP (Sigma-Aldrich, B5900-.5MG), [1,5,10]μM valsartan combos respectively with [0.2,1,2] μM BNP, [10,30,60]mM glutathione (Sigma-Aldrich, G4251-1G), [1,3,5] μM CW-HM12 (Cayman Chemical Company, 19480), [10,20,50] μM salbutamol (Sigma-Aldrich, S8260-25MG), [5,10,25] μM marimistat (Sigma-Aldrich, M2699-5Mg), [1,5,10] μM galunisertib (Selleck Chemicals, S2230), [12.5,25,50] μM fasudil (Sigma-Aldrich, CDS021620-10MG), [10,25,50]μM SB203580 (Sigma-Aldrich, S8307-1MG), [1,5,10] mg/mL pirfenidone (Sigma-Aldrich, P2116-10MG), [5,10,20] μM defactinib (MedChem Express, HY-12289A), [5,10,20] μM WH-4-023 (Sigma-Aldrich, SML1334-5MG), and 20 μM LY294002 (Selleck Chemicals, S1105).

Techniques: Single Cell, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Logic-based mechanistic machine learning on high-content images reveals how drugs differentially regulate cardiac fibroblasts

doi: 10.1101/2023.03.01.530599

Figure Lengend Snippet: A) Images of human cardiac fibroblasts treated with baseline control stimulus, TGFβ, or TGFβ + WH-4-023. B) Quantification of Actin Long Angular Second Moment (ASM), a measure of F-actin uniformity and reduced stress fibers based on images in panel A. C) Regression coefficients from the LogiMML mechanistic machine learning model that predicts network modules that regulate actin long ASM. D) Knockdown sensitivity analysis predicting individual proteins that regulate actin long ASM in the TGFβ+WH-4-023 signaling context. E) Signaling schematic for WH-4-023 effect on actin long ASM, derived from sensitivity analysis in panel D. F) Human cardiac fibroblasts treated with PI3K inhibitor LY294002 or baseline control stimulus, measuring F-actin and procollagen expression. G) Quantification of long actin Angular Second Moment (measure of F-actin uniformity), F-actin integrated intensity, and Procollagen I integrated intensity. *p≤0.05 ANOVA with Tukey’s post-hoc in panel B, and *p≤0.05 Student’s T-test in panel G.

Article Snippet: The drugs with their respective concentrations are as follows: [0.25,1,2] μg/ml of anakinra (Kineret, SOBI Inc.), [1,5,10] μM valsartan (Sigma-Aldrich, SML0142-10MG), [0.2,1,2] μM BNP (Sigma-Aldrich, B5900-.5MG), [1,5,10]μM valsartan combos respectively with [0.2,1,2] μM BNP, [10,30,60]mM glutathione (Sigma-Aldrich, G4251-1G), [1,3,5] μM CW-HM12 (Cayman Chemical Company, 19480), [10,20,50] μM salbutamol (Sigma-Aldrich, S8260-25MG), [5,10,25] μM marimistat (Sigma-Aldrich, M2699-5Mg), [1,5,10] μM galunisertib (Selleck Chemicals, S2230), [12.5,25,50] μM fasudil (Sigma-Aldrich, CDS021620-10MG), [10,25,50]μM SB203580 (Sigma-Aldrich, S8307-1MG), [1,5,10] mg/mL pirfenidone (Sigma-Aldrich, P2116-10MG), [5,10,20] μM defactinib (MedChem Express, HY-12289A), [5,10,20] μM WH-4-023 (Sigma-Aldrich, SML1334-5MG), and 20 μM LY294002 (Selleck Chemicals, S1105).

Techniques: Control, Knockdown, Derivative Assay, Expressing

Journal: International Journal of Proteomics

Article Title: iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

doi: 10.1155/2013/581862

Figure Lengend Snippet: Expression analysis of five cellular proteins and comparison of different quantitation methods. Lysates from Ctrl (noninfected cells) and HAdV-B3/C5-infected A549 cells (biological duplicates) were used for Western blot analyses. Alpha-tubulin was used as a loading control for the normalization. Western blot analyses and quantitative comparisons were performed for Gal1 (LEG1_HUMAN) (a), Gal3 (LEG3_HUMAN) (b), AG2 (AGR2_HUAMN) (c), and CD98 (4F2_HUMAN) (d). nq: no quantitation value was obtained.

Article Snippet: The mouse monoclonal antibody with specificity for anterior gradient protein 2 (AG-2, cat: sc-101211) and the goat polyclonal antibody with specificity for 4F2 cell-surface antigen heavy chain (CD98, cat: sc-31251) were purchased from Santa Cruz Biotechnology Inc.

Techniques: Expressing, Comparison, Quantitation Assay, Infection, Western Blot, Control