![Coculture with BMSCs does not protect against NVP-BEZ235–induced WM cell cytotoxicity. (A) BCWM.1 cells were cultured with NVP-BEZ235 (6.25-100nM) for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed using the [3H]-thymidine uptake assay. (B) BCWM.1 cells were cultured with either NVP-BEZ235 (5-100nM) alone or in presence of BMSCs for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, p-mTOR, anti-raptor, anti-rictor, and anti–α-tubulin antibodies. (C) Adhesion assay with BCWM.1 in the presence or absence of NVP-BEZ235 (5-50nM). BCWM.1 demonstrated increased adhesion in FN-coated wells compared with control. NVP-BEZ235 inhibited adhesion in FN-coated wells in a dose-dependent manner (P < .05). All data represent mean (± SD) of triplicate experiments. Adhesion of BCWM.1 cell to primary BMSCs. BCWM.1 demonstrated increased adhesion in BMSC-coated wells compared with control. NVP-BEZ235 inhibited adhesion to BMSCs in a dose-dependent manner (P < .02). All data represent mean (± SD) of triplicate experiments. (D) Transwell migration assay. SDF-1 (30nM) was placed in the lower chambers, and migration was determined after 3 hours. Transwell migration assay showing inhibition of migration of BCWM.1 in the presence of NVP-BEZ235 (5-50nM). SDF-1 (30nM) was placed in the lower chambers and induced migration compared with control with no SDF-1. NVP-BEZ235 inhibited SDF-1–induced migration in WM cells (P < .05). (E) BCWM.1 cells were cultured with NVP-BEZ235 (6.25-100nM) for 4 hours. Whole-cell lysates were subjected to Western blotting using anti–p-focal adhesion kinase, anti–p-paxillin, anti–p-cofilin, and anti–α-tubulin antibodies. (F) In vivo flow cytometry. Calcein red/orange–labeled cells treated with 20nM NVP-BEZ235 and calcein green/AM–labeled, untreated cells were injected in the tail vein of 2 BALB/c mice. Cells were counted every 5 minutes for 60 minutes. Fluorescence signal was detected on an appropriate artery in the ear and digitized for analysis with Matlab software (*P < .05).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/3/10.1182_blood-2009-07-235747/4/zh89990947450007.jpeg?Expires=1722120404&Signature=lHFD10SMKfJecOWwLFZTAVRWOA5f9RfNeBLSfocSA7fsCEoxtd3cuI4ohzMqU-sCByOiCg7tHBvI62~hgD-x1s9HbDYG6VjbYavOExvyzBPdBJRedLE~lJcjGUlfQRoq1F418cHGCHsseG3hFctrzPqJJhz0u49cYw9EreeSzsyv7JNo-mWcnr-B2ZTkWU1szb8kkGY6Bi5jc~8mdVt83yjggDKAc3eKpULypFCqjLmcr-awU6eDPQkdLOhs3jGoZm7duPmNefj1n6FdLjdmMKKtOc6suANHzesGefwhM5eG5WDlL94g962p9hgKcdDpg-JOjSaPv-NyFZrgPL84FQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Coculture with BMSCs does not protect against NVP-BEZ235–induced WM cell cytotoxicity. (A) BCWM.1 cells were cultured with NVP-BEZ235 (6.25-100nM) for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed using the [3H]-thymidine uptake assay. (B) BCWM.1 cells were cultured with either NVP-BEZ235 (5-100nM) alone or in presence of BMSCs for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, p-mTOR, anti-raptor, anti-rictor, and anti–α-tubulin antibodies. (C) Adhesion assay with BCWM.1 in the presence or absence of NVP-BEZ235 (5-50nM). BCWM.1 demonstrated increased adhesion in FN-coated wells compared with control. NVP-BEZ235 inhibited adhesion in FN-coated wells in a dose-dependent manner (P < .05). All data represent mean (± SD) of triplicate experiments. Adhesion of BCWM.1 cell to primary BMSCs. BCWM.1 demonstrated increased adhesion in BMSC-coated wells compared with control. NVP-BEZ235 inhibited adhesion to BMSCs in a dose-dependent manner (P < .02). All data represent mean (± SD) of triplicate experiments. (D) Transwell migration assay. SDF-1 (30nM) was placed in the lower chambers, and migration was determined after 3 hours. Transwell migration assay showing inhibition of migration of BCWM.1 in the presence of NVP-BEZ235 (5-50nM). SDF-1 (30nM) was placed in the lower chambers and induced migration compared with control with no SDF-1. NVP-BEZ235 inhibited SDF-1–induced migration in WM cells (P < .05). (E) BCWM.1 cells were cultured with NVP-BEZ235 (6.25-100nM) for 4 hours. Whole-cell lysates were subjected to Western blotting using anti–p-focal adhesion kinase, anti–p-paxillin, anti–p-cofilin, and anti–α-tubulin antibodies. (F) In vivo flow cytometry. Calcein red/orange–labeled cells treated with 20nM NVP-BEZ235 and calcein green/AM–labeled, untreated cells were injected in the tail vein of 2 BALB/c mice. Cells were counted every 5 minutes for 60 minutes. Fluorescence signal was detected on an appropriate artery in the ear and digitized for analysis with Matlab software (*P < .05).
