Secretion and functional activity of MM-derived SHH. (A) RT-PCR showing the levels of mRNA for DISP in highly purified CD138⁺ primary MM cells from 10 patients with MM (Pt MM) and 6 MMCLs. (B) Enzyme-linked immunosorbent assay (ELISA) showing the levels of secreted SHH in culture media of highly purified CD19⁺ B cells (Pt B cell; n = 4) or CD138⁺ primary MM cells (Pt MM; n = 10) from MM patients’ BM aspirates and 6 MMCLs. (C-D) Hh signaling detected as luciferase activity in HEK293 cells induced by coculture with MM cells (left panels) or culture with MM cell culture supernatants (SNs; right panels). (E-F) Hh signaling detected as levels of mRNA examined by real-time PCR for GLI1 (left panels) and PTCH1 (right panels) in MM cells induced by MM-derived SHH in the presence or absence of cyclopamine (10 μM) or SHH-neutralizing antibody (aSHH; 5 μg/mL) for 48 hours. (G) Luciferase reporter gene assay shows the reduced pGL3-GLI activity in ^SHH–ARP-1 or ^SHH–CAG cells. (H) ELISA shows the reduced SHH secretion in the supernatant of 24-hour cultured ^DISP1–ARP-1 or ^DISP1–CAG cells. *P < .05; **P < .01. ctl, control.