Zhu et al. Journal of Experimental & Clinical Cancer Research 2012, 31:31 http://www.jeccr.com/content/31/1/31 Page 2 of 7 Methods Cell line and cell culture Breast cancer cell lines MDA-MB-435, MDA-MB-231 and MDA-MB-468 and B cell line Ramos were pur- chased from Cell Bank of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MDA-MB-435 cells and Ramos cells were cul- tured 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 from Abcam Inc (Cambridge, MA). Anti-Lamin B, anti-NF-kappa B p65 antibodies and don- key 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-1a antibodies and goat anti-rabbit secondary antibodies were obtained from Cell Signaling (Beverly, MA) Anti-b-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, 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 (pro- moterless) luciferase vector and pRL-SV40 plasmid were obtained from Promega (San Francisco, California, USA). API-1, SB 202190, PX 12 and Caffeic acid phe- nethyl ester (CAPE) were from Tocris (Bristol, 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 pur- chased from Qiagen (Shanghai, China). Migration assay Cell migration assay were performed in a double cham- ber transwell (Corning) with polycarbonate membranes (8.0 μm pore size). 8 × 104 cells were added to the upper chamber, treated with or without specific antago- nists. Different concentrations of BLyS were added to the lower chamber. 1% FBS was used as a negative con- trol. After incubation at 37 for 8 h in hypoxic or nor- moxic conditions, migrated cells were stained and counted in five randomly selected fields. Quantitative real-time PCR Total RNA was extracted using a Trizol reagent (Invi- trogen Corporation, Grand Island, NY, USA) and was reversed to cDNA using RevertAid™ first strand cDNA Synthesis Kit according to the manufacturer’s instruc- tions. All primers were synthesized by Sangon Biotech (Shanghai, China) or TAKARA (Dalian, China). The pri- mers used in Q-PCR are listed as follow: BLyS (Gen- Bank, 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); b-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). The gene levels in the resultant cDNAs were examined using the detection system (TAKARA) with SYBR-green as fluorescent dye enabling real time detection of PCR products according to the manufacturer’s protocol. The relative expression levels of the genes were determined against b-actin levels in the samples. Western blotting analysis Total cell lysates were prepared in RIPA buffer supple- mented with protease inhibitors. The proteins were frac- tionated by 8%-12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane (Bio-Rad). The membranes were probed with primary antibodies and then probed with relative secondary antibody. b- actin was used as a loading control. Immunofluorescence For BLyS and its three receptors staining in cells, MDA- MB-435, MDA-MB-231, MDA-MB-468 cells and Ramos cells were seeded on coverslips and cultured in 5% CO2 incubator. At 12 h after subculture, the plate with Ramos cells was centrifuged at 1, 000 rpm for 10 min and all the cells were fixed in 4% paraformadehyde for 10 min, washed and incubated with anti-BLyS antibody, anti-BAFF-R antibody, anti-BCMA antibody and anti- TACI antibody (1:100 dilution in 1% BSA-PBS). The cells were then incubated with relative FITC-conjugated secondary antibody (1:1000 dilution in 1% BSA-PBS) for 1 h at room temperature and with Hoechst 33342 for 30 minutes. The processed cells were mounted and fluorescence microscopy images were taken from five random fields in each slide using an inverted micro- scope (Olympus IX 71, Japan). Plasmid construction, transient transfection and luciferase assays pGL3-Basic luciferase vector, a plasmid of luciferase- reporter for human BLyS promoter (GenBank,