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SUPPLEMENTARY METHODS
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Cells
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ATL-derived HTLV-1-infected T-cell lines ED40515(-)
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expression, an HTLV-1-transformed T-cell line MT-2
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Epstein-Barr virus-transformed B-cell line LCL-Kan were used. These cells were
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cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100
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U/ml of penicillin and 100 g/ml of streptomycin. Peripheral blood samples were
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donated from four patients with acute ATL, after they provided their written informed
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consent. Peripheral blood mononuclear cells (PBMC) were isolated from the samples
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using Ficoll-paque PLUS (GE Healthcare UK, Ltd., Buckinghamshire, United
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Kingdom) density gradient centrifugation and then stored frozen in liquid nitrogen until
12
use. This study was approved by the ethics committee of Tokyo Medical and Dental
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University.
1
2
and MT-12 lacking a viral
producing HTLV-1, and an
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15
Primers
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The forward and reverse primers at various regions of the HTLV-1 gene were purchased
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from Greiner Japan (Tokyo). When designing the primers, we used the nucleotide
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sequence and number according to the J02029 clone containing the complete proviral
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genome 3. The orientation, location and nucleotide number starting from the 5’ end of
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the R region of 5’ HTLV-1 LTR as well as the primers used in this study are summarized
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in Supplementary Table 1.
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In addition to HTLV-1 primers, the following primers were used to quantify mRNA
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of NIK (forward, 5’- ACAACGAGGGTGTCCTGCTC - 3’ ; reverse, 5’- TCAGCTCCT
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CTGCCCGAAA - 3’), ICAM1 (forward, 5’- CTGGACTCCAGAACGGGTG - 3’ ;
1
1
reverse,
2
GGAGGGCAGAATCATCACG - 3’ ; reverse, 5’- TCGATTGGATGGCAGTAGCT -
3
3’), CD25 (forward, 5’- CCACACGCCACATTCAAAGC - 3’ ; reverse, 5’-
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TGTGTTCCGAGTGGCAGAGC
5
CCGCACCTCCACTCCATCC - 3’ ; reverse, 5’- ACATCAGCACCCAAGGACACC -
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3’), PKR (forward, 5’- CCTGTCCTCTGGTTCTTTTGCT - 3’ ; reverse, 5’-
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GATGATTCAGAAGCGAGTGTGC
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GTGAAGGTCGGAGTCAACGGATTTGGTCGT
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TGATTTTGGAGGGATCTCGCTCCTGGAAGA - 3’).
5’-
GTAGGGTAAGGTTCTTGCCCAC
-
3’),
-
3’),
-
3’),
VEGF
IκB-α
and
-
(forward,
GAPDH
3’
(forward,
;
5’-
5’-
(forward,
5’-
reverse,
5’-
10
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Differential RT-PCR for sense and anti-sense RNA
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Total RNA extracted from cells using Isogen (Nippon Gene, Tokyo, Japan) was treated
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with DNase (Ambion, Austin, TX), and the aliquots (1 g) were subjected to
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quantitative RT-PCR (RT-qPCR) using real-time qPCR (LightCycler, Roche
15
Diagnostics). Anti-sense or sense RNAs corresponding to various HTLV-1 regions were
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differentially reverse transcribed with forward or reverse primers alone for each primer
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set, respectively, using the Rever Tra AceR qPCR RT Kit (ToYoBo, Osaka, Japan), and
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the resulting cDNA was subjected to qPCR with the same primer set using the
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THUNDERBIRDTM qPCR Mix (ToYoBo). The primer sets used were RexRe-Fw2 and
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RexRe-Rev for the R region, U5-fw and U5-rev for the U5 region, gag-fw and gag-rev
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for the Gag region, pX2 and pX3 for the pX region, and LTR 6-28 and U3-R1 for the
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U3 region. In order to analyze the HBZ mRNA, oligo-dT primers were used for RT
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followed by qPCR with the spliced HBZ-specific primer sets (HBZ 98-Fw and HBZ
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193-217R) 4. For the evaluation of mRNAs other than HTLV-1 transcripts, oligo-dT
2
1
primers were used for RT. The thermal cycle consisted of an initial denaturation step
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at 98℃ for 30 seconds, followed by 40 cycles of denaturation at 95℃ for five seconds,
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annealing at 60℃ for 10 seconds, extension at 72℃ for 30 seconds and detection at
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86 ℃ for two seconds. The RNA copies were standardized according to the
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simultaneously quantified GAPDH mRNA level of the same total RNA sample.
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5’-Rapid amplification of cDNA ends (RACE)
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In order to determine the 5′ end of the anti-sense transcripts, RACE was performed
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using the SMART RACE cDNA amplification kit (Clontech, US), according to the
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manufacturer's instructions. First-strand cDNA was synthesized by RT from 1 μg of
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total RNA of ED40515(-) cells or PBMCs obtained from acute ATL patients using
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forward primers specific for the HTLV-1 R region (RexRe Fw2) or U5 region（627-651
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Fw）. The first cycle of PCR amplification was performed using the AdvantageR 2 PCR
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Kit (Clontech, US) with the Universal Primer A Mix provided in the kit and either the R
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region-specific primer（473-497 Fw）or U5 region-specific primer (666-689 Fw),
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respectively. Nested amplification was performed using the Nested Universal primer A
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in the kit and either the U5-specific primer (577-601 Fw) or U5-specific primer
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(724-747 Fw), respectively. The PCR products were visualized on ethidium bromide
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staining following electrophoresis, and the DNA fragments in major bands excised from
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the gel were cloned into pGEM-Teasy (Promega) vectors. The nucleotide sequences of
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the cloned products were determined using the ABI sequence system (Perkin-Elmer
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Applied Biosystems) with the M13 Fw primer located in the cloning plasmid, according
23
to the manufacturer's instructions.
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1
2
RNA interference
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Two
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UGGAACUUUCGAUCUGUAACGGCGC,
5
UGAGAGUGCUAUAGGAUGGGCUGUC), four siRNAs targeting anti-sense R
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sequences (si-R409-433: CCGGCGUGGAUGGCGGCCUCAGGUA, si-R475-499:
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ACUUACCUAGACGGCGGACGCAGUU,
si-R500-524:
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GGCCCGGUCUCGACCUGAGCUUUAA,
si-R571-595:
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AGCAAGCAGGGUCAGGCAAAGCGUG), siRNA targeting anti-sense U3 sequences
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(si-U3 136: UGCUGAGCUGCCCUCCGGGGGGAGA), or siRNA targeting HBZ
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(si-HBZ611: GGCGAAACAGCAUAGUGCUAGGAAA) were obtained through the
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custom service (Life Technologies, Tokyo, Japan) and used either alone or in a mixture,
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together with appropriate combinations of control siRNAs (si-control GC31:
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UAAUGUAUUGGAACGCAUA, si-control GC47: AGGUAGUGUAAUCGCCUUG,
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si-control GC68 ： UGCGCUAGGCCUCGGUUGC). Transfection of siRNA was
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performed via electroporation into 1x 106 cells in 100 l of Ingenio Electroporation
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Solution (Mirus, Madison, WI) containing 30 nM of siRNA with the AmaxaTM program
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T-6 or T-19 for ED40515(-) and T-14 for ED40515(-) reporter cells on the
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NucleofectorTM (Lonza, Basel, Switzerland).
siRNAs
targeting
anti-sense
U5
sequences
(si-LTR-628:
si-LTR-733:
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Immunoblotting
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Cells were cultured in the presence or absence of 10 M of Z-Leu-Leu-Leu-H (MG132)
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(Peptide Institute, Osaka, Japan) for three hours and dissolved in RIPA buffer (50 mM
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Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton X100, 0.5% Sodium
4
1
Deoxycholate), including protease inhibitor cocktail (Roche Diagnostics, Basel,
2
Switzerland) and PhosSTOP (Roche Diagnostics, Basel, Switzerland), for one hour on
3
ice. The cell lysates were cleared via centrifugation and denatured with SDS sample
4
buffer (Thermo Scientific, Rockford, IL) and 2.5% 2-mercaptethanol (Sigma-Aldrich,
5
St. Louis, MD) at 70°C for 15 minutes, after which 20 μg of proteins were
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electrophoresed on polyacrylamide gel (Oriental Instruments CO., LTD, Kanagawa,
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Japan) and then transferred to PVDF membranes (ATTO, Tokyo, Japan). The membrane
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was blocked with Block Ace (DS Pharma Biomedical Co., Ltd, Osaka, Japan) for one
9
hour at room temperature and reacted with primary antibodies overnight, followed by
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exposure to anti-rabbit IgG, HRP-linked antibodies (Cell Signaling, Danvers, MA) or
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anti-mouse IgG, HRP-linked antibodies (Cell Signaling, Danvers, MA) as secondary
12
antibodies. The reacted bands were visualized using enhanced chemiluminescence with
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NovelR ECL (Invitrogen, Carlsbad, CA) and analyzed on the Image Quant mini LAS
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4000 (GE Healthcare).
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Antibodies and reagents
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Mouse anti-PKR antibodies (Santa Cruz, Dallas, Texas) and rabbit anti-phospho-PKR
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(Abcam, Cambridge, UK), anti-NIK, anti-NFκB2 p100/p52, anti-phospho-NFκB2 p100
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(Ser866/870), anti-phospho-NFκB p65 and anti-β-Actin (Cell signaling, Danvers, MA)
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antibodies were used as primary antibodies for the immunoblotting assays.
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Phycoerythrin-conjugated anti-human IL-2R alpha monoclonal antibodies and
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phycoerythrin-conjugated murine IgG2a isotype control (R＆D systems, Minneapolis,
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MN) were used for flow cytometry on the FACSCalibur system (Becton Dickinson, San
24
Jose, CA). A chemical PKR inhibitor (an imidazolo-oxindole derivative C16,
5
1
C13H8N4OS; Calbiochem)
2
Calbiochem) were dissolved in DMSO and added at a concentration of 100-500 nM in
3
culture.
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and its negative control inhibitor (C15H8Cl3NO;
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5
Reporter assay
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ED40515(-) reporter cells were prepared using a Cignal Lenti-NF-B reporter Luc Kit
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(Qiagen, Duesseldorf, Germany) and Cignal Lenti thymidine kinase (TK)-Renilla
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control (Qiagen). The luciferase assays were conducted with the Luciferase or Renilla
9
luciferase assay systems (Promega, Madison, WI) on cell lysates in Renilla luciferase
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lysis buffer (Promega). The relative NF-B activity was calculated as the ratio of the
11
firefly luciferase activity to the Renilla luciferase activity in the same sample.
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13
Statistics
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The unpaired t-test was performed for statistical significance, and p values less than
15
0.05 were considered significant.
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17
References
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1.
Maeda M, Shimizu A, Ikuta K, Okamoto H, Kashihara M, Uchiyama T, et al.
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Origin of human T-lymphotrophic virus I-positive T cell lines in adult T cell
20
leukemia. Analysis of T cell receptor gene rearrangement. The Journal of
21
experimental medicine 1985 Dec 1; 162(6): 2169-2174.
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2.
Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, et al.
Type C virus particles in a cord T-cell line derived by co-cultivating normal
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1
human cord leukocytes and human leukaemic T cells. Nature 1981 Dec 24;
2
294(5843): 770-771.
3
4
3.
Seiki M, Hattori S, Hirayama Y, Yoshida M. Human adult T-cell leukemia virus:
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complete nucleotide sequence of the provirus genome integrated in leukemia
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cell DNA. Proc Natl Acad Sci U S A 1983 Jun; 80(12): 3618-3622.
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4.
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Satou Y, Yasunaga J, Yoshida M, Matsuoka M. HTLV-I basic leucine zipper
factor gene mRNA supports proliferation of adult T cell leukemia cells. Proc
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Natl Acad Sci U S A 2006 Jan 17; 103(3): 720-725.
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Jammi NV, Whitby LR, Beal PA. Small molecule inhibitors of the
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