Figure 4.

Anti-osteolytic activity of SL-401. (a) CD14⁺ monocytes isolated from MM patient PBMCs samples (n =5) were stimulated with hu-RANKL (40 ng/ml) and hu-M-CSF (25 ng/ml) for 3 weeks, with analysis of cells for CD123 (IL-3Rα) and CD14 at days 1, day 9 and day 18. (b) Monocytes derived from MM patient PBMCs were cultured with M-CSF and RANKL for 2 weeks to promote OCL differentiation, in the presence or absence of indicated concentrations of SL-401. OCLs were analyzed for tartrate-resistant acid phosphatase (TRAP) staining. Micrograph shows OCL formation in untreated vs SL-401-treated cells (black arrows indicate multi-nucleated OCL). (c) Monocytes derived from MM patient PBMCs were cultured with M-CSF and RANKL for 2 weeks to promote OCL differentiation, in the presence or absence of indicated concentrations of SL-401. TRAP activity was measured in supernatants of OCL culture by using a colorimetric substrate. A dose-dependent decrease in absorbance at 540 nm is indicative of the effect of SL-401 on OCL formation. SL-401 reduced number of TRAP⁺ cells vs control culture (P<0.003). (d) OCL bone resorption was analyzed during OCL differentiation using OsteoAssay plates (dentine discs), in the presence or absence of SL-401. The captured images (d) of resorbed surface area were analyzed using NIH ImageJ software, and data (e) is presented as percentage of resorbed surfaces in untreated vs SL-401-treated cells. Black arrows in ‘d’ indicate multi-nucleated OCL, and blue arrows indicate resorbed pit on the OsteoAssay surface. (f) MM BM-derived OBLs were stimulated with ascorbic acid (0.05 mg/ml), β-glycerophosphate (2.16 mg/ml) and 10 nM of dexamethasone for 3 weeks in the presence or absence of indicated concentrations of SL-401, followed by assessment of bone mineralization (calcium deposits) using Alizarin Red staining (mean ±s.d.; n =4; P<0.05).