Reagents and cell culture conditions
The cell lines used in the current study include HL-60 (Human promyelocytic leukemia), U-937 (Human leukemic monocyte lymphoma), THP-1 (Human acute monocytic leukemia), MEG-01 (megakaryoblastic leukemia) and MOLT-4 (Human acute lymphoblastic leukemia). They were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to the manufacturer's instructions. Tan IIA (National Institute for the Control of Pharmaceutical and Biological Products, P.R.China) was dissolved in DMSO solution (0.01%). The stock concentration of Tan IIA was 200 μg/mL. L-sulforaphane (L-SFN) was purchased from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd.
Cytotoxicity assay for various cancer cell lines
Cell viability was evaluated by the 3-[4, 5-dimethylthiazol-2, 5]-diphenyl tetrazolium bromide (MTT) assay in triplicate . For the experiments comparing the five cell lines, each cell line (2.5 × 105 cells/well) was cultured in the recommended medium in 96-well plates for 24 h. The culture medium was then removed, and the cells were treated with 0.1% DMSO as vehicle control or Tan IIA at 30 μg/mL. At the end of the cultivation, 20 μl of MTT working solution was added to the wells, which were incubated for an additional 4 h at 37°C. Finally, the absorbance of each well was measured using a micro-titer plate reader (TECAN, Vienna, Austria) at 570 nm. For the dose- and time-dependent experiments, the U-937 cells were treated with Tan IIA at concentrations of 1, 2, 3, 5 and 10 μg/mL for 0, 12, 24, 36 and 48 h, respectively.
Detection of Tan IIA-induced apoptosis in U-937 cells using Annexin V and Caspase-3 assays
The assays were performed as described previously . Briefly, the U-937 cells were maintained in DMEM medium plus 10% fetal bovine serum. Tan IIA (3 μg/mL) was added and the cells were cultured for 12, 24, 36 and 48 h, respectively. The vehicle control groups were treated with 0.1% DMSO. Apoptotic cell death was examined using annexin V-FITC/PI and active caspase-3-PE reagent kits (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Ten thousand events were acquired for each sample and analyzed by flow cytometry using Lysis II software (FACScan; BD Pharmingen).
To determine the expression levels of genome-wide gene transcripts in Tan IIA-treated and control cells, RNA was extracted using RNA extraction kit (Ambion, TX) according to manufacturer's instructions. Five μg of high-quality total RNA per sample was first converted to double-stranded cDNA. Biotin-labelled cRNA was subsequently synthesized on cDNA templates and fragmented prior to hybridization to Affymetrix GeneChip HG-U133 plus 2 slides following the manufacturer's recommendations (Affymetrix, Santa Clara, CA, USA). The integrity of cDNA, cRNA, and fragmented cRNA was assessed by running aliquots on the Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA, USA). Hybridized probes were detected with streptavidin-phycoerythrin and scanned on an Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA, USA).
Microarray data analysis
Microarray images were visually inspected for hybridization artifacts and then analyzed with Affymetrix GeneChip Operating Software (GCOS) according to Affymetrix's guidelines (http://www.affymetrix.com). Affymetrix Microarray Suite MAS 5.0 algorithm was used for intensity estimation and data normalization. The cell intensity files (.cel) of all arrays were also analyzed using dChip software , which employs the invariant set normalization method to normalize the arrays. In addition, the PM-only model was used to detect the less-abundant genes in a more sensitive fashion [16, 17]. Functional annotation of genes was performed at the NETAFFX Analysis Center. MAPPFinder  was used to find all pathway maps containing the significantly differentially expressed genes (SDEGs). The GeneChip data have been submitted to NCBI's Gene Expression Omnibus with accession number GSE33358.
Real-time quantitative Polymerase Chain Reaction (RT-qPCR)
U-937 cells were treated with Tan IIA at 3 μg/mL and cultured for 1, 2, 4, 6, 12, 24 and 36 h. Total cellular RNA was extracted using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). The cDNAs were synthesized using M-MLV reverse transcriptase with oligo-d (T) 15 primers according to the manufacturer's instructions (Promega, Madison, WI, USA). The primers used to detect the expression of target genes are listed in Additional file 1, Table S1. ABL was used as internal control. RT-qPCR was performed with SYBR-Green I intercalating dye (Bio-Rad, Hercules, CA, USA) using a MyiQ Single Color Real-time PCR Detection System (Bio-Rad). The following PCR program was used: an initial denaturation at 95°C for 5 min, 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 20 s. The RT-qPCR data were analyzed using the MyiQ software (Bio-Rad). All experiments were independently repeated three times.
Enzyme-linked immunosorbent assay (ELISA)
U-937 cells were treated with Tan IIA at different concentrations of 0, 1, 3, 6 and 18 μg/mL for 12 h and harvested for ELISA. The expression level of CCL2 was determined using an ELISA kit (R & D, Minneapolis, MN, USA) following the manufacturer's instructions. A monoclonal antibody specific for CCL2 was pre-coated onto a microplate. Standards and samples were added to the wells. After washing away the unbound substances, enzyme-linked polyclonal antibodies specific for CCL2 were added to the wells. Subsequently, substrates were added. The colors developed were in proportion to the amount of CCL2 bound on the wells. The optical density of each well was measured using a microreader (DNATECH MR 5000) at a wavelength of 450 nm.
U-937 Colony-Forming Unit (CFU) Assay
The assay was performed as described previously . Colony-forming unit-U937 were cultured in methylcellulose (1%) supplemented with fetal calf serum (FCS, 30%), 1% BSA, 0.1 mM ß-mercaptoethanol, and with or without Tan IIA (2 ug/ml), PXR inhibitor L-sulforaphane (L-SFN) (1 uM) . U937 cells (1 × 103 cells/mL) were seeded in triplicate and incubated for 3 days. The colony was considered as a cluster of 10 or more cells. Colonies were scored blindly.
All experiments were performed in triplicate. The results were expressed as mean ± SD. Correlation analyses were performed using JMP software (version 6, SAS, NC, USA).