The human ATL cell lines, S1T and Su9T01, and the HTLV-1-infected T-cell lines, Oh13T, K3T, F6T, and MT-2, were maintained in Gibco RPMI 1640 supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (all from Thermo Fisher Scientific Inc., Waltham, MA, USA). All cell lines, except MT-2, were established from patients in our laboratory . We purchased MT-2 cells from the Japanese Cancer Research Resources Bank (JCRB1210; Osaka, Japan). We analyzed Tax protein-positive and -negative cell lines to determine the influence of Tax on NOTCH1 protein expression and function, as well as on cell growth. We also examined NOTCH1 protein expression in clones K3T and F6T that produce Tax, and the S1T and Su9T01 clones that do not. We also examined the T-ALL cell lines and JURKAT cells that are without a NOTCH1 mutation. JURKAT cells do have a FBXW7 mutation, which causes higher NOTCH signaling. Moreover, they have a PTEN deletion, resulting in PI3K pathway activation and thus a rescue from GSI [8, 9]. SUP-T1, with t(7;9)(q34;q34.3), resulting in aberrant expression of the NTM and absence of full-length NOTCH1, and HD-Mar, with t (9;14)(q34.3;q11.2), carrying breakpoints located inside HD, resulting in high GSI sensitivity despite NOTCH1 overexpression , were also examined. We used the human Burkitt lymphoma cell line Namalwa and peripheral blood lymphocytes (PBLs) from healthy individuals with or without activation. The SUP-T1, HD-Mar, and Namalwa cell lines were obtained from DSMZ (Department of Human and Animal Cell Cultures, Braunschweig, Germany). Activated PBLs were incubated with 10 U/mL of recombinant human IL-2 (Amgen Biologicals, Thousand Oaks, CA, USA) for 6 days at 37 °C in 95% humidity under a 5% CO2 atmosphere. S1TcTax consisted of cell lines stably transfected with the Tax expression vector pcTax WT that harbors wild-type tax cDNA and the neomycin-resistance gene. Control S1TcNeo harbored a plasmid containing the neomycin resistance gene. S1TcTax05 and S1TcTax10 clones express abundant Tax mRNA . The Ethics Committee and Institutional Review Board of Kagoshima University approved the study, in which healthy persons provided written informed consent to participate.
Proteins were analyzed by western blotting as follows. Cells (4 × 106) were lysed with 50 μL of RIPA buffer comprising 50 μL 2× SDS buffer, 1 μL aprotinin (1 mg/mL), and 5 μL of phenylmethylsulfonyl fluoride (20 ng/mL). Lysates (20 μL) were loaded onto 6% SDS-PAGE gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and blotted onto nitrocellulose membranes (Schleicher Schuell, Dassel, Germany) using a semi-dry technique. Loading was checked using Ponceau dye and an anti-human β-actin antibody (Ab) (Santa Cruz Biotechnology Inc., Dallas, TX, USA). We obtained the following NOTCH1 Abs: mN1A Ab against ANK domain (BD Biosciences, San Diego, CA, USA) and Ab8925 against TM/RAM domain in ICN1 (Abcam, Cambridge, UK). The Ab8925 epitopes require prior exposure to GS and detected GS-cleaved NOTCH1, according to the manufacturer. For accuracy, we performed western blotting analysis three times.
N-[N-(3,5-difluorophenyl)-L-alanyl]-S-phenyl-glycine t-butyl ester (DAPT; Peptides International Inc., Louisville, KY, USA) was dissolved in 10 mM dimethyl sulfoxide (DMSO) stock solution.
MTT reduction assays
Cell lines were cultured for 96 h with the indicated concentrations of DAPT or DMSO. Cell viability was then determined using MTT assays (Sigma-Aldrich Corp., St Louis, MO, USA).
We created libraries using Ion AmpliSeq™ Exome technology (Thermo Fisher Scientific Inc.) and then genotyped amplicons by whole-exome sequencing (WES) using an Ion Proton™ platform (Life Technologies Corp., Carlsbad, CA, USA) as described by the manufacturer. Sequences were aligned against a reference genome (GRCh37/hg19) using TMAP (Thermo Fisher Scientific Inc.), and then genotyped variants were confirmed by Sanger sequencing.
Data are shown as means ± standard error (SE). Statistical significance was determined using Student t-tests. Values with p < 0.05 were considered statistically significant.