Osteocalcin

Immunoblotting was performed as previously described [16 (link)]. Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies to Foxc2, CD31 (Abcam, ab5060, ab182982. Cambridge, UK), Osterix (Santa Cruz Biotechnology, sc-22536), and Osteocalcin (ThermoFisher Scientific, PA1-511A). β-actin (Sigma-Aldrich, A2228) was used as a loading control.

FACS analysis was performed as previously described [24 (link)]. A syringe was used to flush out bone marrow from femurs with phosphate-buffered saline (PBS). After that, femurs were cut into small pieces, which were incubated in collagenase solution (collagenase I and collagenase II mixture) for 25 min at 37 °C. After the incubation, the digested mix was collected and washed with cold PBS three times. The collected cells were filtered through cell strainers (70 µm and 40 µm) and centrifuged at 300× g. A total of 2 mL red blood cell lysis buffer was added to the precipitated cells for 10 min. We collected the cells again and washed them with PBS three times. After this, the cells were ready for staining. The cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or Alexa Fluor 488 (AF-488)-conjugated antibodies against CD31 and VE-cadherin (all from BD biosciences, 550274 and 562243, Franklin Lakes, NJ, USA) and Osteocalcin (ThermoFisher Scientific, 23418, Waltham, MA, USA). Nonspecific fluorochrome- and isotype-matched IgGs (BD Pharmingen, Franklin Lakes, NJ, USA) served as controls. A gating strategy was utilized as we used before [24 (link)]. Forward scatter (FSC) and side scatter (SSC) were used to set up the gates and regions, and cell populations were analyzed using specific markers.

Cell pellets were extracted from alginate gels as described above. The pellets were lysed in RIPA lysis buffer with protease and phosphatase inhibitors (ThermoFisher Scientific), and the protein concentration was determined using the BCA assay. Samples were diluted in Laemmli Sample Buffer (Bio-Rad) to 2.5 µg/µl and 25 µg was loaded in 4-15%, 15-well gels (Bio-Rad). The gels were run for 35 min and the protein was then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% non-fat milk for 1 h then incubated with primary antibodies overnight (TRPV4, Abcam ab39260, 1:300, p38 Santa Cruz Biotech. sc-535, 1:1000, osteocalcin, Alfa Aesar, J65216, 1:250, lamin A/C, Cell Signaling Technology, #2032, 1:500). Secondary antibodies against the primary (Licor, IRDye800CW) were incubated with the blot for 1 h. The blots were imaged using a Licor Odyssey imaging system.