Figure 6. SCD-O cells secrete sclerostin.

(a) Immunoblot of SCD-O total cell lysates revealed increased sclerostin expression at 15 days. Exposure to PTH for 48 hrs reduced sclerostin expression, whereas little to no change was seen following LIV treatment (a). (b) Sclerostin was measured in a 2 d collection of conditioned media. Treatment with PTH and LIV reduced secretion of sclerostin (b). Sclerostin protein production was confirmed by immunocytochemistry. Control nodules stained positively for sclerostin (c) staining, which decreased after 48 hr PTH exposure (d). (e) Axin2 mRNA expression from mdMSCs was determined following exposure to serum free media (SF), conditioned media (CM) from SCD-O cells collected from day 15-17 days, conditioned media from SCD-O cells treated for 48 hrs with PTH followed by 24 hrs of serum free media (CM PTH), or conditioned media from SCD-O cells treated for 4 days with LIV (2 × 20 mins daily, CM LIV). CM (50%) was added to mdMSCs overnight. The next day, mdMSCs were incubated with Wnt10b or PBS control. Addition of Wnt10b to mdMSCs incubated in SF media induced a significant increase in Axin2 expression. The elevated Axin2 response with Wnt10b exposure was ablated following overnight incubation with CM from SCD-O cells, with no significant (ns) increase over cell treated with PBS. SCD-O CM after PTH or LIV resulted in a significant increase in Wnt10b induced Axin2 expression. Blots were cropped for clarity; full-length blots are presented in supplementary figure 1. All Western blots were run under the same experimental conditions.