Cd3 pan t lymphocytes

Five previously characterized NPM‐ALK⁺ T‐cell lymphoma cell lines: Karpas 299, SR‐786, SU‐DHL‐1, SUP‐M2, and DEL (DSMZ, Braunschweig, Germany), were used in the study (Drexler, 2010). The breast cancer cell line MCF7 and neuroblastoma cell line SK‐N‐AS (ATCC, Manassas, VA, USA) were used as positive controls for the expression of NGF and TrkA, respectively. Human peripheral blood CD3⁺ pan‐T lymphocytes were purchased from StemCell Technologies (catalog number: 70024; Vancouver, BC, Canada). The ALK inhibitor ASP3026 (CT‐ASP302; ChemieTek, Indianapolis, IN, USA) was dissolved in DMSO mixed with H₂O and HCl (1 : 1).
Cells were maintained in RPMI‐1640 medium (NPM‐ALK⁺ lymphoma cell lines) or Dulbecco's modified Eagle's medium (MCF7, SK‐N‐AS) (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat‐inactivated fetal bovine serum (FBS), penicillin (100 U·mL⁻¹), streptomycin (100 μg·mL⁻¹), and l‐glutamine (2 mm) (all from Sigma Aldrich, St. Louis, MO, USA). Cell cultures were maintained at 37 °C in humidified air with 5% CO₂. In some experiments, cells were cultured overnight in 0.5–1% FBS. Then, 250 or 500 ng·mL⁻¹ recombinant human β‐NGF (256‐GF‐100/CF; R&D Systems, Minneapolis, MN, USA) was added with or without 5 μg·mL⁻¹ anti‐TrkA‐neutralizing antibody (AF175; R&D Systems).

TLL (Jurkat and MOLT-4) and MF/SS (HuT 78, and H9) cell lines were purchased from American Type Culture Collection (Bethesda, MD). Normal human peripheral blood CD3⁺ pan-T lymphocytes were purchased from StemCell Technologies (Vancouver, BC, Canada; Catalog number: PB009–1F). Cells were maintained in RPMI-1640 medium (HyClone; Thermo Fisher Scientific, Waltham, MA), supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The γ-secretase inhibitor GSI-I (Z-Leu-Leu-Nle-CHO) was purchased from Merck Calbiochem (San Diego, CA). The proteasome inhibitor BTZ, the AKT inhibitor MK-2206, and the p-p38 MAPK inhibitor SB203580 were obtained from Selleckchem (Houston, TX). A concentration-response curve was generated for each cell line as shown in Fig. 3, then, a concentration equivalent to IC₃₀ specific for each cell line was used in the in vitro experiments.