SNX-2112 acts in the BM microenvironment to inhibit angiogenesis and osteoclastogenesis. (A) HUVECs were cultured with or without 125 nM SNX-2112 for 12 hours, and tube formation was assessed using microscopy. VEGF free medium and protein kinase C inhibitor enzaustaurin were used as a negative or positive control for growth inhibition (original magnification ×4). (B) HUVECs were cultured with SNX-2112 (125 nM) for indicated times (left panel) and cultured for 12 hours with indicated doses of SNX-2112 (right panel). Cell lysates were analyzed by immunoblotting. (C,D) Osteoclastogenesis was determined by TRAP assay. PBMNCs were cultured for 3 weeks in the presence of 50 ng/mL of M-CSF and RANKL, with or without SNX-2112. SNX-2112 decreased multinucleated TRAP-positive cells (P < .01). Images were obtained using a Leica DMIL microscope equipped with a 4×, 10×/0.22, and 40×/0.60 numeric aperture objective lens (Leica Microsystems, Wetzlar, Germany) and acquired through IM50 software (Leica Microsystems Imaging Solutions, Cambridge, United Kingdom). Original magnification ×4. (E) PBMNCs were cultured with M-CSF and RANKL for the indicated days, in the presence or absence of 10 nM of SNX-2112. Cell lysates were analyzed by Western blotting.