VICs were seeded on hydrogels (E≃5 kPa, Supplemental Figure 1 ) and treated with either human recombinant annexin A2 (R&D Systems) or cystatin C (R&D Systems) at various concentrations. For inhibition studies, cells were treated with a known p38 MAPK inhibitor, SB203580 (Selleck Chemicals), at the previously optimized concentration of 20 μM 21 (link). After immunostaining (refer to the following section), gel samples were imaged on a glass-bottom 24 well plate (Cellvis) using either an Operetta High-Content Imaging System (Perkin Elmer, Part no. HH12000000) or Nikon Eclipse Ti2-E. Sample images were analyzed by adapting a publicly sourced MATLAB code from GitHub 26 . Myofibroblast activation was assessed by quantifying α-SMA gradient mean intensity values and osteoblast-like differentiation was measured by quantifying RUNX2 nuclear intensity normalized to RUNX2 cell mask intensity. Individual cell values were analyzed for at least 5 images per gel, with a minimum of two gels per condition.
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Annexin A2 and Cystatin C Effects on Myofibroblasts and Osteoblasts
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Corresponding Organization : Sanford Consortium for Regenerative Medicine
Other organizations : University of Colorado Boulder
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Variable analysis
independent variables
- Human recombinant annexin A2 (R&D Systems) at various concentrations
- Cystatin C (R&D Systems) at various concentrations
- SB203580 (Selleck Chemicals), a known p38 MAPK inhibitor, at the previously optimized concentration of 20 μM
- Myofibroblast activation, assessed by quantifying α-SMA gradient mean intensity values
- Osteoblast-like differentiation, measured by quantifying RUNX2 nuclear intensity normalized to RUNX2 cell mask intensity
- Hydrogels with an approximate Young's modulus (E) of 5 kPa
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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