Reagents.Anti-Sclerostin (Scl) antibody (Scl-Ab) and control IgG for the in vivo experiments were generously donated by Eli Lilly and Co (Indianapolis, IN); Scl-Ab (Ab63097) used for immunohistochemistry was from Abcam (Cambridge, MA);Scl-Ab for the in vitro experiments (AB-108-C) and IgG (AF1589) were from R&D Systems (Minneapolis, MN); γ-secretase inhibitor (GSI) XX from Calbiochem (San Diego, CA); Bortezomib (BTZ) from Selleck Chemicals (Houston, TX); Dexamethasone (DEX) from Sigma-Aldrich (St. Louis, MO); and RPMI-1640, penicillin, and streptomycin from Invitrogen Life Technologies (Grand Island, NY).

Cell culture. JJN3 and 5TGM1 cell lines were provided by N. Giuliani (University of Parma, Italy) and maintained as previously described (1).All cell lines were confirmed as mycoplasma-free. Cell lines were authenticated by immunoglobulin production and CD138 expression.

Immunohistochemistry. For static bone histomorphometric analyses, longitudinal sections of the proximal tibiae were stained for hematoxylin and eosin, TRAPase or von Kossa, and counterstained with Toluidine blue as previously described(2). Scl was visualized in deparaffinized sections of decalcified tibiae as described before(1). Briefly, tissue sections were treated with 3% H2O2 to inhibit endogenous peroxidase activity, blocked with serum, and then incubated with goat polyclonal anti-mouse Scl-Ab (1:100). Sections were then incubated with anti-goat biotinylated secondary anti-body followed by avidin conjugated peroxidase (Vectastain Elite ABC Ki t; Vector Laboratories, Burlingame, CA, USA; catalog PK-610 5). Color was developed with a diaminobenzidine substrate chromogen system (DAKO, Carpinteria, CA, USA).Analyses were performed in a blinded fashion.

Statistics. Data were analyzed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA). Data exceeding two standard deviations from the mean were considered as outliers and removed from the analysis. Data of the Sost-/-/Scid in vivoexperiments were analyzed by two-way ANOVA using genotype and multiple myeloma as independent variables. Data of the Scl-Ab treatment in vivoexperiments were analyzed by two-way ANOVA using treatment and multiple myeloma as independent variables. Data of the Scl-Ab treatment in vitroexperiments were analyzed by two-way ANOVA using treatment and anti-MM drugs as independent variables. For all the experiments, when ANOVA detected a significant interaction between the variables or a significant main effect of genotype/treatment, a post hoc test was used to determine the significance of the effect of multiple myeloma within each genotype. All pairwise multiple comparison procedures within the two-way ANOVAtests using the Tukey method were utilized to detect genotype and/or treatment differences. Differences inhuman Kappa light chain levels (only detected in mice bearing JJN3 MM tumors) and in the number/area of osteolytic lesions (only observed in mice bearing MM tumors) were evaluated by t-test. Data were considered significantly different at p ≤ 0.05.

Data availability.The data that support the findings of this study are available from the corresponding author upon reasonable request.

Reference List

(1) Delgado-Calle J, Anderson J, Cregor MD, Hiasa M, Chirgwin JM, Carlesso N, et al. Bidirectional Notch signaling and osteocyte-derived factors in the bone marrow microenvironment promote tumor cell proliferation and bone destruction in multiple myeloma. Cancer Res 2016;76:1089-1100.

(2) Delgado-Calle J, Tu X, Pacheco-Costa R, McAndrews K, Edwards R, Pellegrini G, et al. Control of bone anabolism in response to mechanical loading and PTH by distinct mechanisms downstream of the PTH receptor. J Bone Miner Res 2017;32:522-535.

1