Colorimetric assay kit

Osteogenic differentiation from human iPSCs of control and OI #3 was performed as mentioned above. MSCs were differentiated into osteoblasts as mentioned above for 2 weeks with or without 0.5 mM 4-PBA. Total proteins were extracted from induced osteoblasts in RIPA buffer (Wako, Tokyo, Japan) containing protease and phosphatase inhibitor cocktail (Wako, Tokyo, Japan). After centrifugation (15,000g, 15 min, 4 °C), supernatants were stored at −80 °C. Protein concentration was measured using a colorimetric assay kit (Bio-Rad, Hercules, CA). Proteins were subjected to SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). After blocking, the membrane was incubated with primary antibody against alkaline phosphatase (R&D Systems, Minneapolis, MN #AF2910) diluted 1:200 and HRP-conjugated anti-β-actin antibody (MBL, Nagoya, Japan #PM053–7) diluted 1:5000. After incubation with the secondary antibody, the signals were visualized using ECL system (GE Healthcare, Bucks, UK). Densitometry was quantified with ImageJ 1.50i (National Institutes of Health, Bethesda, MD).

Commercial ELISA kits (eBioscience, San Diego, CA) were used to measure the levels of phosphorylated and total STAT3, according to the manufacturer's instructions. Briefly, cells were lysed in lysis buffer supplied with the kit at room temperature for 20 min. The protein concentration in each sample was then measured using a colorimetric assay kit (Bio-Rad, Hercules, CA) to confirm that the amount of protein was sufficient for further tests. The lysed sample and the positive control (supplied with kit) were then transferred to the coating plate and mixed with an equivalent volume of the antibody cocktail supplied with the kit. The mixture was then incubated in the dark for 2 h. After washing, the detection reagent was added for 30 min. Finally, stop solution was added to each well and the colorimetric reaction was measured at 450 nm. STAT3 activity was expressed as a ratio of the amount of phosphorylated to total STAT3 after normalization against the total protein concentration.