Runx2

Protein expression was demonstrated by Western blotting, following instructions described previously [4 (link)]. Moreover, cell cytoplasm and nucleus proteins were separated using kits from Beyotime. The following antibody dilutions were used: BMAL1 (Proteintech, 1:500), runx2 (CST, 1:1000), alkaline phosphatase (ALP) (R&D, 1:500), gapdh (Proteintech, 1:1000), Lamin B1 (Proteintech, 1:1000), phospho-JNK MAPK (cst, 1:1000), phospho-NF-kB (cst, 1:500), phospho-AKT (cst, 1:1000), phospho-IκBα (cst, 1:500) and phospho-p65 (cst, 1:1000).

Cellular extracts were prepared, as previously described [23 (link)], and immunoblotting was done via standard procedures, according to the manufacturer’s instructions. Adipocyte differentiation markers were identified using antibodies against CCAAT/enhancer binding protein α (C/EBPα, Santa Cruz Biotechnology, Dallas, TX, USA) and peroxisome proliferator-activated receptor γ (PPARγ, Santa Cruz Biotechnology). Osteoblast differentiation markers were detected using antibodies against osteocalcin (Santa Cruz Biotechnology), Runt-related transcription factor 2 (RUNX2, R&D Systems, Minneapolis, MN, USA), and Dickkopf-related protein 1 (DKK1, R&D Systems). The expression of senescence-associated cell cycle inhibitors was determined using antibodies against p16^INK4A and p21^WAF1/CIP1 (BD-Pharmingen, BD Biosciences, San Jose, CA, USA). Tubulin (Sigma Aldrich) was used as an internal control for protein loading. The antibodies were detected with a chemiluminescence detection kit (Amersham Biosciences GE Healthcare Europe, Velizy Villacoublay, France). Western blot quantification was performed in triplicate using Fiji software (Open source) and results were normalized to the Tubulin protein levels.