Fig. 2

Assessment of cellular responses to H₂O₂ using MCF-7 cells. a Western blotting shows efficient depletion of PRDX1 protein in the MCF-7-HyPer-3 cells. b Fluorescence of HyPer-3 (green) in live sgNTC-pool3 and sgPRDX1-pool3 MCF-7 cells after adding exogenous H₂O₂ at final concentration of 100 µM at the indicated time points. c Fluorescence intensities of HyPer-3 in the sgNTC-pool3 and sgPRDX1-pool3 MCF-7 cells untreated and incubated with 100 µM H₂O₂. Green arrow indicates adding of H₂O₂. Experiment was performed twice. d Concentration-dependent cytotoxicity of H₂O₂ produced by GOx in CRISPR/Cas9-engineered MCF-7 cells. Cells were treated with a range of GOx concentrations (0.25; 0.5; 1 mU/ml) for 24 h. Control cells were cultured without any reagent. At the end of treatment, the crystal violet staining (or MTT test, see Suppl. Fig. S3A) was performed and reported as percent growth relative to control. e Determination of cell death by propidium iodide staining followed by flow cytometry analysis. Cells were treated with a range of GOx concentrations (0.5; 1 mU/ml) for 24 h. Control cells were cultured without any reagent. Experiments were performed in duplicates and repeated three times, ***p < 0.001. f Relative fluorescence intensity (log₂ values) of PY1 probe after reaction with H₂O₂ produced by GOx in sgPRDX1 and sgPRDX2 MCF-7 cells compared to controls. Cells treated with 1 mU/ml of GOx were preincubated with catalase (100 μg/ml) for 30 min. After 24 h of GOx treatment 10 μM PY1 dye was added to the medium for 30 min at 37 °C and the read was taken using EnVision reader at the excitation wavelength 514 nm and emission wavelength 550 nm. Each sample was at least triplicated, and data were obtained from three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected (**p < 0.01, ***p < 0.001)