Ppar gamma

ASC (n = 3) and MD-ASC (n = 3) cultured in differentiation medium were fixed in 4% buffered paraformaldehyde for 20 min at room temperature (R.T.). For intracellular staining, fixed cells were permeabilized for 10 min at R.T. with 0.3% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) before exposing them to primary antibodies. Unspecific binding was blocked using 5% BSA in phosphate-buffered saline (PBS) for 1 h at R.T., and cells were incubated overnight at 4 °C to detect Peroxisome Proliferator-Activated Receptor Gamma (PPAR-Gamma) (1:100; Santa Cruz Biotechnology. Inc., Heidelberg, Germany). A488 (Molecular Probe, Invitrogen, Carlsbad, CA, USA) was used as secondary antibodies and was diluted to 1:800 for 1 h at 37 °C. Finally, Vectashield (Vector-Labs, Burlingame, CA, USA) added with 0.1 μg/mL DAPI (Sigma, St Louis, MO, USA) was used as a mounting medium.
Epifluorescence images were acquired using a Leica DMI 6000B microscope (Leica Microsystems, Wetzlar, Germany) at 40× oil immersion (numerical aperture: 1.25). Adobe Photoshop software was utilized to compose and overlay the images and to adjust contrast (Adobe, San Josè, CA, USA). The differentiation potential was calculated by determining the fraction of ASC cells displaying either Oil Red-O or PPAR-Gamma positivity. At least 200 cells were assessed. All experiments were performed in triplicate.

The cellular proteins were extracted using 1 × RIPA buffer from cell pellets and the concentration of cellular proteins was measured using a BCA protein assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% skim milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20), the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies against ALP, OPN, Runx2, Osterix, BMP-2, Smad-1, p38, ERK, JNK, β-actin (1:5000 dilution), phospho-Smad1, phospho-p38, phospho-ERK, phospho-JNK (1:3000 dilution), PPAR gamma, c-Fos, and NFATc1 were used from Santa Cruz Biotechnology (San Diego, CA, USA). Next, the nitrocellulose membranes were primed with secondary antibodies (Anti-Rabbit or Anti-Mouse with horseradish peroxidase). Immunoreactive protein bands were detected using a chemiluminescent reagent (Thermo Fisher Scientific). Relative protein images were captured using Fusion SOLO X (Vilber, Marne-la-Vallée, France).