Journal: PLoS ONE

Article Title: Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK

doi: 10.1371/journal.pone.0183003

Figure Lengend Snippet: Ba/F3 cells were infected with an empty virus (-) and retrovirus expressing NPM-ALK or TEL-JAK2. (A) Whole cell lysates were immunoblotted with an anti-ALK antibody, anti-Flag antibody, anti-JAK2 antibody, anti-phospho-STAT3 antibody, anti-STAT3 antibody, anti-phospho-STAT5 antibody, anti-STAT5 antibody or anti-β-actin antibody. (B) Transduced Ba/F3 cells were incubated with RPMI containing 10% FBS in the absence of IL-3 for 2 days. Viable cell numbers at 24 hr and 48 hr were counted by the Trypan blue exclusion method. Values are the mean ± SD of three independent experiments. (C-F) Transduced Ba/F3 cells were incubated with RPMI containing 10% FBS in the presence of crizotinib (0.25, 0.5, 1 μM) and MMC (0.1, 1, 10 μg/mL) for 24 hr. (C) Cell viability was measured by the WST assay. Values are the mean ± S.D. of four independent experiments. (D) Cells were fixed, treated with propidium iodide, and subjected to a flow cytometric analysis. The percentages of cells in the sub-G1 phase were graphed. Values are the mean ± S.D. of four independent experiments. (E) To evaluate the apoptotic cell death, the chromatin DNA was isolated from cells and subjected to agarose gel electrophoresis. (F) Whole cell lysates were prepared and the phosphorylation of STAT3 and STAT5 was analyzed by immunoblotting. The relative phosphorylation level of STAT3 and STAT5 is shown in the graphs. Values are given as the mean ± SD of three independent experiments. *** P < 0.001 significantly different from control group of Ba/F3 cells expressing NPM-ALK.

Article Snippet: SUDHL-1 cells and Ki-JK cells were cultured in RPMI 1640 medium supplemented with 10% FBS (BioWest), 100 units/ml penicillin (Nacalai Tesque), and 100 μg/ml streptomycin (Nacalai Tesque) with or without IL-3 (2 ng/mL) at 37°C and 5% CO 2 .

Techniques: Infection, Virus, Expressing, Incubation, WST Assay, Isolation, Agarose Gel Electrophoresis, Phospho-proteomics, Western Blot, Control