Recombinant human il 4

CLL cells were cultured at 2 × 10⁶/ml in complete medium as previously described^{23 (link),25 (link)}. For stimulation studies, recombinant human IL-4 (Immunotools, Friesoyte, Germany) was used at 25 ng/ml. In signal transduction studies, CLL cells were pretreated for 2 h with the PI3Kδ inhibitor Idelalisib (CAL-101, 5 µM; Selleck Chemicals, Houston, TX, USA), the PKCδ inhibitor Rottlerin (10 µM; Calbiochem, La Jolla, CA, USA) or DMSO as control, before culture with IL-4 for further 24 h. For neutralization studies, CLL cells were cultured in 96-well plates at 2 × 10⁵/well in 200 µl complete medium containing 60 µg/ml human anti-Jag1 (AF1277) or normal goat IgG control (AB-108-C), all from R&D Systems (Minneapolis, MN, USA).

CD19⁺ B cells were isolated from PBMC of healthy volunteers by negative selection using the B cell purification kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) and following manufacturer’s instructions. Purified B cells (with a purity of more than 95%) were incubated with PI3Kδ inhibitors for 1 h, and stimulated with 10 μg/ml goat F(ab′)2 anti-IgM (Southern Biotech) and 10 ng/ml recombinant human IL-4 (Immunotools, Friesoythe, Germany) for 4 days. [³H] thymidine (1 μCi; Perkin Elmer, Shelton, CT, USA) was added for the last 18 h of culture. Proliferation was assessed by a multiplate beta counter (Perkin Elmer, Shelton, CT, USA).

CLL cells (12 × 10⁶) were resuspended in 100 μl Cell Line Solution Kit V (Lonza Group Ltd, Basel, Switzerland) with ON-TARGETplus SMARTpool small interfering RNA (siRNA) to Notch1 (0.5 μM), Notch2 (0.5 μM) or ON-TARGETplus siCONTROL nontargeting pool as negative control (all from Dharmacon RNA Technologies, Lafayette, CO). In the case of combined Notch1 and Notch2 silencing, 0.25 μM per siRNA was added. Cells were then transfected with the Amaxa Nucleofector II device (program U-013) and cultured for 72 hours in 12-well plates in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen, Milan, Italy). In some experiments, transfected cells were incubated with 25 ng/ml recombinant human IL-4 (Immunotools, Friesoyte, Germany), 50 μM pan-caspase inhibitor z-VAD-fmk or 2.5 μM proteasome inhibitor MG132 (both from Calbiochem, La Jolla, CA). The inhibitors z-VAD-fmk and MG132 were dissolved in DMSO and diluted in complete medium at the used concentrations. DMSO concentrations, which did not exceed 0.005%, did not affect CLL cell responses. IL-4 and z-VAD-fmk were added at the beginning of the 72-hour post transfection culture and maintained until cell collection, whereas MG132 was added during the last 4-hour culture.