Anticancer activity of WT, Q59L, and Q59F l-ASP. (A-H) Two ovarian cancer cell lines (OVCAR-8 and SK-OV-3) and 6 leukemia cell lines (MOLT-4, K562, NALM-6, REH, SR, and CCRF-CEM) were seeded in 96-well plates, incubated for 48 hours, treated with a range of (WT, Q59L, or Q59F) l-ASP concentrations for 48 hours, and finally assayed with CellTiter-Blue using fluorescence excitation at 544 nm and emission at 590 nm. (I-J) MOLT-4 and OVCAR-8 cells were seeded in 96-well plates and incubated for 48 hours, then treated with indicated concentrations of E colil-ASP WT or Q59 mutant for 48 hours. Inhibition of cell viability was measured as in panels A-H. Sham treatment was used as a control. (K) Western blot analysis of ASNS levels in the indicated cells treated with an EC50 dose of l-ASP. (L) Western blot analysis of ASNS levels in OVCAR-8 cells treated with l-ASP mutants. Numbers below the blot represent the relative level of ASNS, which was normalized to the level of the loading control β-actin (set to “1” for the control).
