MDA-MB-435 cells and Ramos cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY) and MDA-MB-231 cells and MDA-MB-468 cells were cultured in L-15 (Gibco, Grand Island, NY), containing

10% fetal bovine serum (Gibco, Grand Island, NY). The cells were used from three to six passages. Materials Anti-human BLyS and anti-human TACI antibodies were obtained from R&D Systems (Minneapolis, MN). Anti-human BAFF-R and anti-human BCMA antibodies were purchased Gefitinib in vitro from Abcam Inc (Cambridge, MA). Anti-Lamin B, anti-NF-kappa B p65 antibodies and donkey anti-goat secondary antibodies were obtained from Santa-Cruz (Santa Cruz, CA). Anti-Akt, anti-p-Akt (Ser 473), anti-p38 MAPK, anti-p-p38 MAPK (Tyr 182), anti-HIF-1α

antibodies and goat anti-rabbit secondary antibodies were obtained from Cell Signaling (Beverly, MA) Anti-β-actin antibody was obtained from Sigma (St. Louis, MO). Goat anti-mouse peroxidase-conjugated antibody was from Sigma (St. Louis, MO). RevertAid™ first strand cDNA Synthesis Kit, selleck kinase inhibitor TurboFect™ in vitro transfection reagent and restriction enzymes Kpn I and Xho I were purchased from Fermentas (Shenzhen, China), Dual-luciferase assay system, pGL3-basic (promoterless) luciferase vector and pRL-SV40 plasmid were obtained from Promega (San Francisco, California, USA). API-1, SB 202190, PX 12 and Caffeic acid phenethyl ester (CAPE) were from Tocris (Bristol, aminophylline UK). Recombinant human BAFF was purchased from R&D system (Minneapolis,

MN). SYBR Premix Ex Taq II and pMD® 18-T Vector were purchased from TAKARA (Dalian, China). DNA purification kit, QIAprep spin miniprep kit and QIAquick gel extraction kit were purchased from Qiagen (Shanghai, China). Migration assay Cell migration assay were performed in a double chamber transwell (Corning) with polycarbonate membranes (8.0 μm pore size). 8 × 104 cells were added to the upper chamber, treated with or without specific antagonists. Different concentrations of BLyS were added to the lower chamber. 1% FBS was used as a negative control. After incubation at 37 for 8 h in hypoxic or normoxic conditions, migrated cells were stained and counted in five randomly selected fields. Quantitative real-time PCR Total RNA was extracted using a Trizol reagent (Invitrogen Corporation, Grand Island, NY, USA) and was reversed to cDNA using RevertAid™ first strand cDNA Synthesis Kit according to the manufacturer’s instructions. All primers were synthesized by Sangon Biotech (Shanghai, China) or TAKARA (Dalian, China). The primers used in Q-PCR are listed as follow: BLyS (GenBank, NM_006573.4) 5′- CGT GCC GTT CAG GGT CCA G-3′ (forward) and 5′-TCG AAA CAA AGT CAC CAG ACT CAA T-3′ (reverse); β-actin (GenBank, AF035119) 5′-CTC CTC CTG AGC GCA AGT ACT C-3′ (forward) and 5′-CGG ACT CGT CAT ACT CCT GCT-3′ (reverse).