D cultureThe human breast cancer cell lines, MCF-7, MDAMB-231, SK-BR-3, ZR-75-1 and the human mammary epithelial cell line, HBL-100, were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). Human multidrug resistant breast cancer cell line, MCF-7/ADR, was obtained from the Cancer Institute of Zhejiang University. HBL-100 was cultured in RPMI-1640 medium (Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 10 fetal bovine serum (FBS, Gibco) as well as 1 penicillin PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28993237 treptomycin (Gibco). The other cells were all cultured in Dulbecco modified Eagle medium (DMEM, Gibco), supplemented with 10 FBS and 1 penicillin treptomycin. Cells were all passaged by trypsinization every 2? days and maintained at 37 in 5 CO2. And all cells were cultured following the guidelines of Laboratory of Xinhua Hospital, affiliated to Shanghai JiaoTong University, School of Medicine, China.Drugs and antibodiesDoxorubicin hydrochloride (ADMh) and Verapamil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). For in vitro studies, they were dissolved in Strokephysiological saline solution (NS, NJCTT Pharmaceutical Co., Ltd, Nangjing, China) to create a stock solution (10 mmol/L), which was stored at -20 . To prepare working solutions, the stock solution was further diluted with culture media to yield the desired ADMh concentration. Primary antibodies against P-gp (goat-anti-rabbit) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies against c-myc, SALL4, BCRP, GAPDH and secondary antibodies (goat-antirabbit) were purchased from Cell Signaling Technology (Danvers, MA, USA).Chen et al. BMC Molecular Biol (2016) 17:Page 3 ofLentivirusmediated RNA interference and transfectionAccording to the gene data of SALL4 (NM-020436) in GenBank, the designed sequence of the short hairpin RNA (shRNA) used to target SALL4 was 5-GCCT TGAAACAAGCCAAGCTA-3. And the sequence of the negative order FPS-ZM1 control shRNA was 5-TTCTCCGAACGTG TCACGT-3.The shRNAs were synthesized and inserted into the pGMLV-SC5 lentivirus core vector containing a cytomegalovirus-driven enhanced green fluorescent protein (GFP) reporter gene. Recombinant lentiviruses expressing SALL4-shRNA (Lv-shSALL4) or negative control shRNA (Lv-shNC) were produced by Genomeditech Co.Ltd (Shanghai, China). The experiment of MCF-7/ ADR cells set up an experimental group (Lv-shSALL4), the negative control group (Lv-shNC) and blank control group (CON). MCF-7/ADR cells were infected with concentrated virus in serum-free medium. The supernatant was replaced with complete culture medium after 24 h. After being transfected for 96 h, mRNA and protein expression of SALL4 in the infected cells was validated by quantitative real-time PCR (qRT-PCR) analysis and western blot assays.Quantitative realtime PCR (qRTPCR)buffer (Beyotime, Shanghai, China) from the treated cells. Protein concentrations were quantified by the BCA protein assay reagent kit (Beyotime) according to the manufacturer’s instructions. Equal quantities of cellular proteins were resolved by SDS-PAGE and then electrophoretically transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). Each membrane was blocked with 5 skim milk and immuneblotted with a primary antibody. After incubation with a secondary antibody, the bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA). GAPDH was used as the loading control.Cell viability assayTotal RNA was extracted from.