Pparγ2

Cells were fixed in 10% formaldehyde, treated with serum-free protein blocking solution (Dako), and then incubated overnight at 4 °C with antibodies against Scx (1:150, Abcam, Rabbit polyclonal), Tnmd (1:150, Santa Cruz Biotechnology, Goat Polyclonal), Runx2 (1:200, Abcam, Rabbit polyclonal), Sox9 (1:200 Santa Cruz Biotechnology, Rabbit polyclonal), PPARγ2 (1:200, Abcam, Rabbit polyclonal), or aggrecan (1:200, Abcam, Rabbit polyclonal). Cells were then rinsed in PBS. For Scx and Tnmd staining, cells were incubated with anti-rabbit Alexafluor 488 (1:1000, Invitrogen) or anti-goat Alexafluor 568 (1:1000, Invitrogen). For the other antibodies, cells were incubated for 30 minutes with anti-rabbit secondary antibody conjugated with horseradish peroxidase (Dako) followed by DAB chromagen (Vector Laboratories) for 2 minutes. Negative control sections were prepared using irrelevant isotype matched primary antibodies (Dako).

APDLSCs and YPDLSCs were washed three times with ice-cold PBS and lysed using RIPA reagent containing 1% PMSF and 1% phosphatase inhibitor cocktail. After centrifugation at 12,000 rpm for 5 min, total protein concentrations were measured using a BCA Protein Assay Kit (Solarbio). Then, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to molecular weight and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% milk for 2 h and then incubated with primary antibodies overnight at 4 °C. Next, the membranes were washed three times with Tris-buffered saline solution with Tween-20 (TBS-T) and incubated with secondary antibodies at room temperature for 1 h. The protein bands were then developed with the use of the Enhanced Chemiluminescence (ECL) Substrate Kit (Millipore), and the densitometry of each band was conducted using ImageJ (National Institutes of Health). The following primary antibodies were used: Runx2 (1:1000, CST), ALP (1:30,000, Abcam), COL1A1 (1:1000, CST), PPARγ2 (1:500, Abcam), CCND3 (1:2000, CST), and GAPDH (1:10,000, Abcam).