The arginase inhibitor Nω−hydroxy−nor−arginine (nor−NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target
Fig 5
Depletion of ARG2 does not induce cell death or compromise the effects of nor−NOHA.
(A) K562 cells were transfected with control, ARG1 or ARG2 siRNA and incubated at 21% or 1% O2 for 48 hours, with or without imatinib (1μM). Levels of ARG2, cleaved PARP and cleaved caspase 3 were detected by western blot on cell lysates. K562 and HL60 cells were transduced with shRNA expressing vector targeting Luc (control) or ARG2, and transduced cells were grown at 21% or 1.5% O2 for 48 hours (B; western) or 72 hours (C; Annexin V/ 7−AAD staining V) before harvesting for the respective assays. (D) Schematic showing the genomic sequence for a portion of the first exon of ARG2. The targeting sequence of two independent sgRNAs are boxed, with the PAM sequence in bold. The translational initiation site (+1) is marked. (E) Characterization of the ARG2-KO clones generated by CRISPR/Cas9 nuclease. The control (Crtl) or ARG2 targeting sgRNA-transduced K562 cells were subjected to puromycin selection to generate single cell clones. The clones were incubated at 21% or 1% O2 for 48 hours, and ARG2 expression was detected by western blot. (F) Arginase activity of the knockout clones was quantified as described for Fig 1D (average of 3 experiments). (G) The control and knockout clones were treated with nor−NOHA (1mM) at 21% or 1.5% O2 for 72 hours, and cell viability was determined by Annexin V/ 7−AAD staining (average of 3 experiments).