Hh activity in MM patient-derived BMSCs. (A) BMSCs isolated from MM patients were stained with Shh-PE–conjugated Ab and analyzed by FACSCanto II flow cytometer; staining is relative to isotype-matched control. As seen, the Shh ligand is variably expressed by BMSCs. (B) MM patient-derived BMSCs were cultured in the presence of control medium or NVP-LDE225 for 48 hours, and proliferation was measured by MTT assay. (C) Immunoblot of protein lysate from MM patient-derived BMSCs fractionated by electrophoresis and stained with anti-Smo, anti-Ptch1, and anti-Gli1 Abs. (D) Immunoblot of protein lysate from MM patient-derived BMSCs fractionated by electrophoresis and stained with anti-Ptch1 Ab, showing no down-regulation of Ptch1 after NVP-LDE225 treatment. (E) MM cell lines were cultured for 48 hours in the presence of control medium or BMSC culture-derived supernatant and treated with NVP-LDE225 at indicated doses; cell viability was assessed by MTT assay. (F) MM cell lines were cultured for 48 hours in the absence or presence of patient-derived BMSCs and treated with NVP-LDE225 at indicated doses; MM cell viability was assessed by CFSE staining.