PMC

Conformation of the recombinant and naturally expressed bomapin is redox-dependent. (A) Purified recombinant bomapin analyzed by SDS-PAGE followed by Coomassie blue staining: lanes 1 and 4, non-reduced bomapin eluted from ion-exchange column in peak 1 and peak 2 (respectively); lanes 2 and 5, DTT-reduced bomapin from peak 1 and peak 2 (respectively); lanes 3 and 6, non-reduced bomapin from peak 1 and peak 2 (respectively) incubated with a 4-fold molar excess of trypsin and then analyzed under reducing conditions. (B) Indirect chromogenic assay to detect inhibitory activity of bomapin. Trypsin alone or trypsin pre-incubated with different amounts of bomapin (at bomapin/trypsin molar ratios as indicated) was mixed with S-2288 chromogenic substrate, and residual trypsin activity was measured at 405 nm in 1 min time intervals. (C) Immuno-localization of naturally expressed bomapin in THP1 cells. The cells were stained with rabbit anti-bomapin antibodies followed by Alexa-Fluor 568-labeled secondary antibody; DNA was stained with DAPI. (D) Bomapin was immunoprecipitated from HL60 cell extract and analyzed by SDS-PAGE followed by western blot: lane 1, non-reduced bomapin; lane 2, DTT-reduced bomapin. (E) In silico homology models of bomapin in its reduced and oxidized forms. The central β-sheet A is shown in blue, the reactive centre loop in red, the CD-loop in yellow, and the two cysteine residues in green.

C395S bomapin mutant, representing reduced form of bomapin, does not enhance proliferation of K562 cells. (A) SDS-PAGE analysis (followed by Coomassie blue staining) of the E. coli expressed and purified recombinant C395S bomapin mutant (lane 1) and the mutant protein incubated with a 4-fold molar excess of trypsin (lane 2). (B) Cellular localization of C395S-bomapin-EGFP in stably transfected K562 cells. (C) Proliferation of the stably transfected multiclonal K562 cells expressing bomapin-EGFP, C395S-bomapin-EGFP, and EGFP alone, as measured by manual cell counting. Higher proliferation of K562 cells expressing wt bomapin, compared to Fig. 2B, is due to higher generation number of the cells that were used in this experiment.

Wt bomapin promotes proliferation of stably-transfected multi-clonal K562 cells. (A) Cellular localization of bomapin-EGFP and EGFP in transfected K562 cells. (B) Proliferation of the K562 cells expressing bomapin-EGFP and EGFP, and the wt K562 cells (seeded at 2 × 10⁴ cell/ml) measured by manual cell counting. The data represent a mean of three independent experiments, each counted in triplicate. (C) Proliferation of the stably transfected K562 cells measured with the WST-1 reagent. (D) Size of nuclei in K562 cells expressing bomapin-EGFP and EGFP. The symbol "..." indicates statistical significance with p < 0.0001 by unpaired t-test. (E) U937 cells were incubated with bomapin-specific antisense (BAS3) and sense (BS) phosphorothioated DNA oligonucleotides (20 nmol/ml); at different time points bomapin was immunoprecipitated with IgY immobilized on NHS-Sepharose and detected with western blot. Western blot of residual amounts of IgY detached from the beads during immunoprecipitation is shown as loading control. (F) U937 cells were seeded at a density of 1 × 10⁴ cells/ml in the absence or the presence of the antisense BAS3 and corresponding sense BS oligonucleotides, and proliferation was measured by manual counting. The data represent the means of three independent experiments, each counted in triplicate. (G) Proliferation of the HT-1080 cells expressing bomapin-EGFP and EGFP (seeded at 2 × 10⁴ cell/ml) measured by manual cell counting. The data represent a mean of three independent experiments.

Bomapin enhances cell apoptosis following growth factors withdrawal. K562 cells and the cells expressing bomapin-EGFP or EGFP were incubated in the presence or the absence of serum in the media. At different time points, cells were mixed with trypan blue and dead cells were quantified by manual counting (A), or the cells were incubated with annexin-PE-Cys5, and annexin-labelled cells were quantified under fluorescence microscope with excitation and emission wavelengths 488 nm and 670 nm, respectively (B); (C) Progression of cell cycle in bomapin-EGFP and EGFP-expressing K562 cells following serum withdrawal. Percentage of G0/G1, S, and G2/M phases were calculated by deconvolution of DNA content histograms; ns - insignificant; ".." indicates statistical significance with p < 0.05. (D) K562 cells expressing bomapin-EGFP or EGFP were incubated in serum-containing media (lanes 1) or in media without serum (lanes 2) for 48 h. Then, cell extracts were analyzed by western blot with monoclonal antibodies against lamins-A/C and rabbit antibodies against cleaved PARP as apoptotic markers. Western blot for β-actin in the same gel is shown as loading control.