Fig. 5

Heterologously expressed recombinant wild-type ADI protein exhibited the same cytostatic phenotype as F-IEX. (a) SDS-PAGE gels analysis of purified proteins. Left gel: ADI WT protein after two steps of purification on Superdex75 (GF75) and Resource Q (IEX) columns (three consecutive fractions collected after IEX separation are shown). Middle gel: Catalytically inert mutant of ADI – C400A. Right gel: Direct comparison of F-IEX and heterologously expressed recombinant ADI WT protein. The red rectangles show the bands corresponding to the calculated size of respective ADI variants (ADI WT or C400A). (b) Representative graphs of confluence changes of HT-29 (top) and HCT-116 (bottom) cells over time (0-72 h) after ADI (0.66% v/v), F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10mM HEPES buffer) treatment with or without the addition of 5 mM L-arginine (ARG). The proliferation of cells was monitored using the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point presents a mean +/- SD. (c) Growth rate constant of data presented in b) (n = 3 independent experiments). (d) Confluence changes of HT-29 (left) and HCT116 (right) cells over time (0-72 h) after ADI (0.66% v/v), vehicle (same as in b) and C400A catalytic mutant (0.66% v/v) treatment. The proliferation of cells was monitored using the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point presents a mean +/- SD. (n = 3 independent experiments). (e) Table with enzymatic activity values of F-IEX, heterologously expressed recombinant ADI WT protein, and ADI C400A catalytic mutant (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test)