Phospho erk1 2

Recombinant human IL-26 protein (MBS1362134) was purchased from MyBiosource (San Diego, CA, USA). Palmitate (P0500), metformin (D150959), methylthiazolyldiphenyl-tetrazolium bromide (MTT) (M5655), and bovine serum albumin (BSA) (A7030) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Antibodies against cyclooxygenase-2 (COX-2; 1:1000; 35-8200) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), while those against collagen type II (COL-II; 1:1000; ab34712) and matrix metalloproteinase-1 (MMP-1) (1:1000; ab134184) were purchased from Abcam (Cambridge, MA, USA), and those against IL-6 (1:1000; #12153), Erk1/2 (1:1000; #4695), phospho-Erk1/2 (1:1000; #9101), phospho-c-Jun (1:1000, #3270), phospho-p38 (1:1000, #9211), phospho-JNK (1:1000, #9251), STAT1 (1:1000, #14994), phospho-STAT1 (1:1000, #9131), STAT3 (1:1000, #9139), phospho-STAT3 (1:1000, #9131), histone H3 (1:2000, #9715), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; #5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The rBMSCs were plated in a six-well plate and cultured until they reached 90% confluency. The cells were induced with osteogenesis or adipogenesis culture medium and incubated in the presence of 150 mg/L of SAA for 7 days. The proteins were subsequently collected after treatment with the combination of cell lysis buffer, phosphatase inhibitor, proteinase inhibitor, their concentration was determined with bicinchoninic acid protein assay kit (Cell Signalling Technology, Shanghai, China). Equal amounts of proteins (20 μg) were separated on SDS-PAGE gels by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with a blocking buffer for 1 h and incubated with the corresponding primary antibody at room temperature. After washing thrice with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated polyclonal goat antibodies, ß-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal references. Extracellular signal-regulated kinase (ERK)-1/2, phospho-ERK1/2, glycogen synthase kinase 3 beta (GSK3β) and phospho-GSK3βantibodies were purchased from Cell Signalling Technology (Shanghai, China), while ß-catenin antibodies were obtained from Abcam (United Kingdom). Peroxisome proliferator-activated receptor gamma (PPARγ) and GAPDH antibodies were supplied by Servicebio (Wuhan, China).