1 mL of fresh StemPro Chondrogenesis media (Thermo Fisher Scientific, Waltham, MA, cat# A10071-01) waddedded to the pellet.
500,000 to 1 × 106 mMSCs were collected and transferred into amber eppendorf tubes (New England Biologicals, Ipswich, MA, cat# B9000).
Next day, growth media was changed to 2 mL of StemPro Osteogenisis Differentiation media (Thermo Fisher Scientific, Waltham, MA, cat# A10072-01).
We mainly used WIT medium (Stemgent, Cambridge, MA, cat no# 00-0045-500) supplemented with 10 μM of p160 ROCK inhibitor Y-27632 dihydrochloride (Selleckchem, Houston, TX) in this study.
The following day, media was changed StemPro Adipogenesis Differentiation media (Thermo Fisher Scientific, Waltham, MA, cat# A10070 01) and differentiation media was changed every 2 3 days for 14 21 days.
Actin was detected with a mouse monoclonal antibody (Millipore; Billerica, MA; cat# MAB 1501).
Rabbit anti-WSTF antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA: Cat No. 2152) and Bethyl Laboratories (Montgomery, TX, USA: Cat No. A300-446A).
Levels of H3K36me3 in set2Δ were examined by probing with anti-Histone H3 (tri methyl K36) antibody (Abcam, Cambridge, MA, USA, cat# ab9050), and GAPDH antibody (Santa Cruz Biotechnology, Dallas, TX, USA, FL-335, cat. sc-25778) was used to visualize the loading control proteins. sc-25778
Neuroblastoma cells (7 × 10) were plated for 2 days in the 'maintenance medium', as follows: hepatocyte defined medium, serum free (BD Biocoat, Bedford, MA, USA, cat. no. 05449), Human EGF (epidermal growth factor) 5 μg/ml (BD Biosciences, Bedford, MA, USA, cat. no. 354052); 2 mM L-Glutamine (Euroclone, Pero (MI), Italy, cat.
For every 50 mg of mouse tissue, 1 ml of 1 × RIPA cell lysis buffer (Cell Signaling, Beverly, MA, USA, Cat. No. 9806) including 1 × protease inhibitors (Calbiochem, EMD Serono, Inc., Rockland, MA, USA, Cat. No. 539134) was added followed by homogenization on ice in a 1.5-ml microcentrifuge tube using a tissue homogenizer (Omni International, Kennesaw, GA, USA, Model TH-115) at high speed for 30 s.
The stained slides were subsequently incubated for 1 h at room temperature with the goat anti‐rabbit IgG Dylight 594 secondary antibody (Thermo Scientific, Waltham, MA, USA, cat. #35560) at 1 100 dilution and mounted with the VECTASHIELD mounting medium with 4'6‐diamidino‐2‐phenylindole (DAPI Vector Laboratorieses, Burlingame, CA, USA, cat# H‐1200).
