SDF-1–induced adhesion and migration in MM cells were inhibited by PCI-32765, and Btk directly regulates MM cell survival. (A) MM cells were preincubated with PCI-32765 (100nM, +) or control media; the drug was then washed out before stimulation with SDF-1 for 2 and 5 minutes. Lysates were immunoprecipitated (IP) with anti-Btk, and the IPs probed with anti-pBtk. Total cell lysates were further probed with indicated phopho-specific Abs and anti–α-tubulin as a loading control. (B) PCI-32765 was added to patient MM cells in overnight coculture with BMSCs. Images were taken using a Leica DFC300FX and Leica IM50 Image Manager (original magnification ×100). (C) CD138⁺ patient MM cells were treated with PCI-32765 and allowed to migrate in the absence (−) or presence of SDF-1 (200 ng/mL) in transwells coated with 1 μg/mL sVCAM-1. Bars represent the mean ± SD of triplicates. (D) MM1R cells were transduced with control GFP or shBtk lentiviruses; cell lysates were then prepared for immunoblotting. MM1R cells pretreated with (PCI) or without (−) PCI-32765 served as controls. (E) Three days after lentivirus infection, MM1R cells were labeled with calcein-AM and adherence to BMSCs was assayed in 2 hours. Four days after lentiviral transduction, cell images (F, original magnification ×400) were taken, and cell viability as well as caspase 3/caspase 7 activity (G) were determined.