Results are presented as mean S.E. and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN- and/or TNF- levels, or inhibitors of Erk1/2 or S6K1 L-Ascorbyl 6-palmitate may be a new approach to prevent BAFF-induced aggressive B-cell malignancies. from our group . Rapamycin was purchased from ALEXIS (San Diego, CA, USA), whereas U0126 was from Sigma (St. Louis, MO, USA). RPMI 1640 Medium was from Gibco (Rockville, MD, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). CellTiter 96? AQueous One Solution Cell Proliferation Assay kit was provided by Promega (Madison, WI, USA). Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit was from BD Biosciences (San Diego, CA, USA). Enhanced chemiluminescence solution was from Millipore (Billerica, MA, USA). Other chemicals used in this work are of analytical grade and were obtained from Sigma and local commercial sources. 2.2. Cell culture Neoplastic B-lymphoid (Raji) cell line (American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin in a humidified incubator of 5% CO2 at 37C. 2.3. Lentiviral shRNA cloning and contamination of cells Lentiviral shRNA to Erk1/2, S6K1, S6K1/Erk1/2 and green fluorescence protein (GFP) (for control) were constructed and infected as described previously [44, 45]. 2.4. MTS assay for cell viability and live cell counting by trypan blue exclusion Raji cells, or Raji cells infected with lentiviral shRNAs to S6K1, Erk1/2 and GFP, respectively, were seeded in 96-well plates (3104 cells/well, for cell viability assay) or 24-well plates (3105 cells/well, for trypan blue exclusion) and cultured for overnight in humidified incubator of 5% CO2 at 37C. Next day, cells were treated with hsBAFF (0C0.25 g/ml), IL-2 (0C100 ng/ml), IL-4 (0C100 ng/ml), IFN- (0C100 ng/ml) or TNF- (0C100 ng/ml) for 48 h, or treated with/without hsBAFF (0.25 g/ml) in the presence or absence of IL-2 (5 and/or 50 ng/ml), IL-4 (5 and/or 25 ng/ml), IFN- (10 and/or 100 ng/ml) or TNF- (5 and/or 50 ng/ml) for 48 h, or pre-incubated with/without U0126 (5 M) for 1 h or rapamycin (0.2 g/ml) for 2 h and then treated with/without hsBAFF (0.25 g/ml) in the presence or absence of IL-2 (50 ng/ml), IL-4 (25 ng/ml), IFN- (100 ng/ml) and/or TNF- (50 ng/ml) for 48 h, with 3C6 replicates of each treatment. Then, cell viability, post incubation with MTS reagent (one solution reagent) (20 l/well) for 4 h, was assayed by monitoring the optical density (OD) at 490 nm using a Synergy? 2 Multi-function Microplate Reader (Bio-Tek Instruments, Winooski, Vermont, USA). Live cells were recorded by counting viable L-Ascorbyl 6-palmitate cells using trypan blue exclusion. 2.5. Cell proliferation analysis and flow cytometry Raji cells were seeded at density of 3105 cells/well (for cell proliferation assay) and 2106 cells/well (for flow cytometry) in 24-well and 6-well plates, respectively. Next day, cells were treated with hsBAFF (0C0.25 g/ml) for 48 h. Subsequently, the number of proliferative cells was counted under a Coulter Counter (Beckman Coulter, Fullerton, CA, USA), and the ratios of live cells were monitored by a FACS Vantage SE flow cytometer (Beton Dickinson, California, USA) using Annexin-V-FITC/PI Apoptosis Detection kit. 2.6. Western blot analysis Raji cells, or Raji cells infected with lentiviral AMLCR1 shRNAs to S6K1, Erk1/2, S6K1/Erk1/2 and GFP, respectively were seeded in 6-well plate (2 106 cells/well) and cultured overnight in humidified incubator of 5% CO2 at 37C. Next day, L-Ascorbyl 6-palmitate cells were treated with hsBAFF (0C0.25 g/ml) for 12 h, or treated with/without hsBAFF (0.25 g/ml) in the presence or absence of IL-2 (5 and/or 50 ng/ml), IL-4 (5 and/or 25 ng/ml), IFN- (10 and/or 100 ng/ml) or TNF- (5 and/or 50 ng/ml) for 12 h, or pre-incubated with/without U0126 (5 M) for 1 h or rapamycin (0.2 g/ml) for 2 h and then treated with/without hsBAFF (0.25 g/ml) in the presence or absence of IL-2 (50 ng/ml), IL-4 (25 ng/ml), IFN- (100 ng/ml) and/or TNF- (50 ng/ml) for 12 h. Afterwards, total cell lysates were subjected to Western blotting as described previously . The antibodies to phospho-Erk1/2 (Thr202/Tyr204), phosphor-S6K1 (Thr389) and phospho-S6 ribosomal protein (Ser235/236) were from Cell Signaling Technology (Beverly, MA, USA), whereas the antibodies to Erk2, S6K1, S6 ribosomal protein and -actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). G oat anti-rabbit IgG-horseradish peroxidase (HRP), goat.
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