Fig. 5: c-Abl is indispensable for the attenuation of cellular ROS levels by HAX-1.

A SH-SY5Y cells transfected with HAX-1 or HAX-1(Q190X) mutant were treated with 1 mM H₂O₂ for 2 h. ROS levels were analyzed by DCFH-DA staining and flow cytometry. The mean frequencies of DCF fluorescence signal intensity were calculated as the mean±S.D. of three independent experiments. n.s., not significant; *p < 0.05, ***p < 0.001, Student’s t test. B MCF-7 scramble, c-Abl/Arg knockdown, HAX-1 knockdown or c-Abl/Arg/HAX-1 triple-gene knockdown cell lines were pretreated with 30 mM 3-AT for 1 h and 1 mM H₂O₂ for 2 h as indicated and stained using DCFH-DA. The fluorescence signal intensity of DCF was detected by flow cytometry on a BD Biosciences FACSCalibur. The mean frequencies of DCF fluorescence signal intensity were calculated as the mean±S.D. of three independent experiments. n.s., not significant; *p < 0.01, **p < 0.01, Student’s t test. C MCF-7 scramble cells, c-Abl/Arg knockdown cells overexpressing HAX-1, and HAX-1 knockdown cells overexpressing c-Abl/Arg were treated with 1 mM H₂O₂ for 2 h and stained using DCFH-DA. The fluorescence signal intensity of DCF was detected by flow cytometry on a BD Biosciences FACSCalibur. The mean frequencies of DCF fluorescence signal intensity were calculated as the mean±S.D. of three independent experiments. n.s., not significant; **p < 0.01, Student’s t test. D MCF-7 scrambled and MCF-7/siHAX-1 cells were treated with 10 μM STI571 for 18 h. Alterations in mitochondrial membrane potential were determined by the ratio of JC-1_red/JC-1_green staining and represented as a ratio of the MCF-7 scramble cells. The mean frequencies of JC-1_red and JC-1_green were calculated as the mean±S.D. of three independent experiments. n.s., not significant; ***p < 0.001, Student’s t test. E MCF-7 scrambled and MCF-7/siHAX-1 cells with or without 10 μM STI571 for 18 h were treated with 1 mM H₂O₂ for 3 h and analyzed by flow cytometry using FITC-Annexin V (FITC-ANV) and propidium iodide (PI) staining. The percentage of cell death (early and late apoptosis) was identified as ANV⁺PI⁺ on a BD Biosciences FACS Calibur. The mean frequencies of apoptotic cells were calculated as the mean±S.D. of three independent experiments. n.s., not significant; *p < 0.05, Student’s t test. F MCF-7 scramble, c-Abl/Arg knockdown, HAX-1 knockdown or c-Abl/Arg/HAX-1 triple-gene knockdown cell lines were analyzed by flow cytometry using FITC-Annexin V (FITC-ANV) and propidium iodide (PI) staining. The percentage of cells undergoing early apoptosis was identified as ANV⁺PI⁻ on a BD Biosciences FACS Calibur. G The mean frequencies of apoptotic cells were calculated as the mean±S.D. of three independent experiments. n.s., not significant; **p < 0.01, Student’s t test.