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. 2012 Jan 10;7(1):e28395. doi: 10.1371/journal.pone.0028395

Figure 1. Schematic representation of the primary structure and functional domains of apoptin, its cytotoxic potency and inhibition of Bcr-Abl phosphorylation.

Figure 1

(A) The SH3 binding domain is merged within NLS1 (amino acids, aa: 82–88). A pictorial representation of apoptin sequences (UniProtKB/Swiss-Prot entry P54094), LRS = Lecine-Rich Sequence, NLS = Nuclear-Localization Signal, NES = Nuclear Export Signal. (B) Cytotoxic activity of apoptin on Bcr-Abl positive 32Dp210 cells: 32Dp210 were grown in 96-well plates (104 cells per well). Cells (in triplicates for each treatment) were treated with 1 µM Tat-apoptin, and Tat-GFP (negative control), or Imatinib for 0, 4, 8, 12, 18 and 24 h periods respectively. The percentage of viable cells, as assessed by MTT assay indicates that apoptin and Imatinib are both toxic to 32Dp210 cells, and that apoptin's cytotoxic effect favorably compares to that of imatinib. Results are expressed as a percent of cell survival (mean ± SD). (C) Apoptin inhibits Bcr-Abl phosphorylation: K562 and 32Dp210 cells were treated with 1 µM Tat-apoptin, Tat-GFP (negative control) or 1 µM imatinib (positive control). Cells were then harvested after 16 hrs and cell lysates were prepared. Representative Western blots show the expression levels of total and phosphorylated Bcr-Abl; equal loading was checked by the loading control, eIF4E. The upper panel of bands shows the expression of K562 cells and the lower panel shows the expression of 32Dp210 cells. Lanes from the left: (1) no-treatment control cell, (2) Tat-GFP treated cell, (3) imatinib treated cell, and (4) Tat-apoptin treated cell respectively in both cell lines. (D) For quantitation, band intensities from immunoblots were scanned by Image Quant software (version 5.2, Molecular Dynamics®). During quantitation, the imatinib expression data was omitted in order to enable visualization of the apoptin effect with greater clarity. Bcr-Abl phosphorylation was significantly inhibited by apoptin. The quantitation data were normalized to the loading control (eIF4E) and expressed as a ratio of phosphorylated to the total Bcr-Abl and presented as mean ± SEM of three independent experiments.