Supplementary methods RNA isolation, cdna synthesis, and quantitative real-time PCR. Total RNAs were extracted from cells by using an RNeasy Mini Kit (QIAGEN) and then reverse-transcribed using the SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative real-time PCR was performed on a Life Technologies ViiA7 Real-time PCR system using the Fast SYBR Green Master Mix (Life Technologies) and specific primer sets for the GAPDH gene (sense: 5 -GCG GCA CGT CAG ATC CA-3 ; antisense: 5 -CAT GGC CTT CCG TGT TTC CTA-3 ) and the CCL3 gene (sense: 5 -GCT GAC AAG CTC ACC CTC TGT-3 ; antisense: 5 -GGC AGT GGT GGA GAC CTT CA-3 ). Relative expression of the CCL3 gene was analyzed by the ΔΔCt method using the Ct value of the GAPDH gene. Flow cytometry. Isolated leukocytes were stained with various combinations of fluorescent dye-conjugated Abs. For intracellular CCL3 and MPO staining, leukocytes were incubated in serum-free S-Clone SF-03 medium (Sanko Junyaku) supplemented with 0.1 % GolgiStop reagent (BD Biosciences) for 4 h. Subsequently, intracellular CCL3 and MPO were stained with PE-conjugated anti-ccl3 Ab and biotin-conjugated anti-mpo Ab, respectively, and incubated with APC and APC-Cy7-conjugated streptavidin, respectively, with the help of an Intracellular Cytokine Staining Starter Kit (BD Biosciences). Intranuclear Ki67 was stained sequentially with biotin-conjugated anti-ki67 Ab and with APC-conjugated streptavidin using the Foxp3/Transcription Factor Buffer Set (ebioscience). Expression of each molecule was determined using a FACSCantoII (BD Biosciences) and analyzed with FlowJo software (Tree Star). 1
In situ hybridization (ISH) assessment of the BM biopsy specimens from patients with CML. For this study, 4-μm sections were prepared from paraffin-embedded tissue and attached to positively charged glass slides. Double-color staining of mrna was conducted using the QuantiGene ViewRNA ISH kit (Panomics Inc.), according to the manufacturer s instruction. Deparaffinized tissue samples were incubated with a pretreatment solution followed by protease digestion. Human CCL3- and ENPP3-specific probes were designed and synthesized by Panomics/Affymetrix. Probe sets and amplifier molecules were hybridized to each pair of oligonucleotides. After the unbound probes were removed by washing, the samples were incubated with alkaline phosphatase to break down the fast red and fast blue substrates to form precipitates. Images of target mrna were acquired using a DP21 microscopic camera (Olympus). Effects of imatinib on cell viability and CCL3 expression in a human basophilic CML cell line, KU812. KU812 cells (2 10 5 /ml) were incubated at the indicated concentrations of imatinib for 24 h and its cell viability was determined by using the cell counting kit-8 (Dojindo Co. Ltd). The ratios of cell viability were determined by comparing the OD value in the absence of imatinib. At the same time, intracellular CCL3 expression was determined gating on ENPP3- and Fc R1-positive cells by using a flow cytometer., Determination of CCL3 expression in total BM cells of CML patients. BM biopsy samples were obtained from 10 CML patients, who were newly diagnosed at Juntendo University Hospital, three times; prior to, or 3 months or 6 months after the initiation of dasatinib treatment. Total RNAs were extracted and were subjected to quantification of 2
the copy number of BCR-ABL gene according to the previously described method. 1 Simultaneously, total RNAs were subjected to quantitative real-time PCR with Life Technologies ViiA7 Real-time PCR system using Thunderbird SYBR qpcr Mix (TOYOBO), and the specific primer sets for GAPDH gene (sense: 5 -aga gac cct cac tgc tg-3 ; antisense: 5 -aga ttc agt gtg gtg gg-3 ) and CCL3 gene 2 (sense: 5 -cca gtt ctc tgc atc act tgc t-3 ; antisense: 5 -ctg ctc gtc tca aag tag tca gct a-3 ). Relative expression of the CCL3 gene was analyzed by the ΔΔCt method using the Ct value of the GAPDH gene. This study was conducted in accordance with the Declaration of Helsinki, and the study design was approved by the Ethics Committee of the Juntendo University (Registration No. 2012195). 3
List of Abs. Antigen Clone Reactivity Host Company CD4 RM4-5 Mouse Rat TONBO Biosciences CD8 53-6.7 Mouse Rat TONBO Biosciences CD11b M1/70 Mouse Rat TONBO Biosciences CD16/32 2.4G2 Mouse Rat BD Biosciences CD19 1D3 Mouse Rat BD Biosciences CD34 RAM34 Mouse Rat ebioscience CD45.1 A20 Mouse Rat TONBO Biosciences CD45.2 104 Mouse Rat TONBO Biosciences CD45R/B220 RA3-6B2 Mouse Rat TONBO Biosciences CD48 HN48-1 Mouse Rat ebioscience CD49b DX5 Mouse Rat BioLegend CD117/c-kit ACK2 Mouse Rat TONBO Biosciences CD150/SLAM TC15-12F12.2 Mouse Rat BioLegend CD200R3 Ba13 Mouse Rat BioLegend FcεR1 MAR-1 Mouse Hamster ebioscience FcεR1 AMR-37 Human Mouse ebioscience ENPP3 NP4D6 Human Mouse BioLegend Ki67 SolA15 Mouse Rat ebioscience Lineage cocktail Mouse Rat BD Biosciences Ly-6A/E/Sca-1 D7 Mouse Rat ebioscience Ly-6G/Gr-1 RB6-8C5 Mouse Rat TONBO Biosciences Ly6G 1A8 Mouse Rat TONBO Biosciences MIP-1α/CCL3 39624 Mouse Rat R & D Systems MIP-1α/CCL3 93342 Human Mouse R & D Systems Myeloperoxidase 2D4 Rat, Mouse Mouse Abcam TCR-β chain H57-597 Mouse Hamster TONBO Biosciences TER-119 TER-119 Mouse Rat TONBO Biosciences Isotype-matched control IgGs for individual rat monoclonal Abs and control mouse IgG were purchased from BD Biosciences. 4
Supplementary references 1. Yoshida C, Fletcher L, Ohashi K, et al. Harmonization of molecular monitoring of chronic myeloid leukemia therapy in Japan. Int J Clin Oncol. 2012;17(6):584-589. 2. Zhang B, Ho YW, Huang Q, et al. Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell. 2012;21(4):577-592. 5
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