Rhopg

PASMCs were stimulated with rhOPG (50 ng ml⁻¹) (R&D systems), alongside quiesced cells (negative control) for 10 and 60 min, before lysing. Cell lysates were mixed with sample buffer (Life Technologies, Carlsbad, CA, USA) and sample reducing agent (Life Technologies), denatured by heating and subjected to gel electrophoresis. The membranes were then incubated with primary antibodies against phospho-CDK4, phospho-HSP27, total mTOR, phospho-mTOR (1:500) and GAPDH (1:1000) (Cell Signalling Technology), CDK5 (1:500) (Abcam), or β-actin (1:1000) (Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were then incubated with anti-Rabbit IRDye 800CW and anti-Mouse IRDye 800CW (Li-COR, Lincoln, NE, USA) and signal detection and band density quantification was performed using the LiCOR Odyssey SA system.

To assess resorption activity of OCLs, RAW 264.7 cells (5×10⁴ cells/well) were plated in calcium phosphate-coated 24-well plates [Osteoclast Activity Assay Substrate (OAAS); Cosmo Bio, Tokyo, Japan] in the presence of RANKL (50 ng/ml) and SDF-1α (20 ng / ml). To test the effect of Th17 cells on OCL activity, Th17 cells (10⁴ cells/well) were added with or without recombinant human OPG (rhOPG, 100 ng/ml, R&D Systems). To test the effect of T-CM on the Th17 cell-OCL interaction, T-CM (20 μl) or OPG knocked-down T-CM was added to OCL cultures. Exogenous rhOPG supplementation (100 ng/ml) in the OPG knocked-down T-CM treatment group was also performed. Non-differentiated CD4+ T cells or a Th17-skewing cytokine cocktail (IL-6, IL-23, and TGF-β) was added to confirm their potential effect on OCL activity. After 6 days of culture, medium was removed, plates were washed with PBS, and cells were incubated with 5% sodium hypochlorite solution for 10 min to detach cells. After washing with PBS three times, plates were dried completely and resorption areas were observed by contrast microscopy. Pit areas were measured using ImageJ software (https://imagej.nih.gov/ij/).