Figure 4: CHK1 regulates nuclear H₂O₂ levels.

(A) CHK1 depletion increases steady-state nuclear H₂O₂ levels in a NAC-dependent manner. Nuclear H₂O₂ levels were measured in K562 co-expressing the indicated sgRNAs and nuclear HyPer7. (B-C) Re-introduction of CHK1 restores nuclear H₂O₂ levels. H₂O₂ levels were determined by nuclear HyPer7 (B) and levels of the indicated proteins by immunoblot (C) following reintroduction of CHK1 into K562 cells depleted of CHK1. (D-E) CHK1 inhibition increases nuclear H₂O₂ in an antioxidant-dependent manner as measured by HyPer7 (D) and roGFP2-ORP1 (E). (F) NAC treatment partially rescues CHK1i cytotoxicity. Relative proliferation was determined after 96 hrs by measuring cellular ATP concentrations. (G) NAC protects cells from CHK1i mediated DNA damage. Immunofluorescence analysis of H2AX•S139 staining (left) and quantification (right). (H) Nuclear H₂O₂ increases CHK1i cytotoxicity to block cell proliferation. K562 cells stably expressing nuclear localized DAAO were pretreated with L-Ala or D-Ala prior to treatment with CHK1i. Relative proliferation was determined as described in (F). Scale bar=10 μm. Data are represented as mean ± SEM. ***P < 0.0001. Statistical significance was determined by Student’s t-test (two-tailed, unpaired).