Figure 5.
ROS mediate sensitization of Erw-ASNase–treated MM cells to Kar. (A) U266 cells were incubated or not with NAC (5 mM) for 2 hours and treated, in the presence or absence, with Erw-ASNase (0.5 U/mL), Kar (2.5 nM), or both. Cell death was assessed by flow cytometry using annexin V (AV) and propidium iodide (PI) double staining after 48 hours. One representative experiment of 3 is shown. (B) Western blot showing that NAC pretreatment (5 mM) rescues proapoptotic features triggered by Erw-ASNase treatment on Kar-exposed U266 cells. Cleavage of PARP1, caspase-3, caspase-9, NRF2, IRF4, γ-H2A.X, and GAPDH (loading control) was assessed. One representative western blot of 3 is shown. (C) qPCR analysis for oxidative stress–induced genes in U266 cells treated with Erw-ASNase (0.5 U/mL), Kar (2.5 nM), or both. The dashed red line indicates the reference level of DMSO-treated cells. (D) MitoSOX staining on U266 cells upon Erw-ASNase treatment (0.5 U/mL), with or without Kar (5 nM) treatment, was evaluated by flow cytometry 28 hours posttreatment. All data are mean ± SD of 3 independent experiments. (E) U266 cells were incubated or not with NAC (5 mM) for 2 hours and then treated, in the presence or absence of NAC, with Erw-ASNase (0.5 U/mL), Kar (3 nM), or both. Mitochondrial superoxide levels were detected by immunofluorescence 24 hours later (left panels). Signals from MitoSOX staining were quantitated with ImageJ software and normalized to control (right panel). One representative experiment of 3 is shown. Magnification ×20. (F) Cell death of mitochondria-targeted catalase (mitoCat) and empty vector–overexpressing LP1 cells treated with the indicated stimuli (0.5 U/mL Erw-ASNase and 3 nM Kar). Cell death was assessed by flow cytometry using annexin V (AV) and propidium iodide (PI) double staining after 48 hours. The percentage of early apoptotic cells (AV+/PI−) are shown as white columns; late apoptotic cells (AV+/PI+) are shown as gray columns. One representative experiment of 3 is shown. *P < .05, **P < .01, ***P < .001, Student t test.

ROS mediate sensitization of Erw-ASNase–treated MM cells to Kar. (A) U266 cells were incubated or not with NAC (5 mM) for 2 hours and treated, in the presence or absence, with Erw-ASNase (0.5 U/mL), Kar (2.5 nM), or both. Cell death was assessed by flow cytometry using annexin V (AV) and propidium iodide (PI) double staining after 48 hours. One representative experiment of 3 is shown. (B) Western blot showing that NAC pretreatment (5 mM) rescues proapoptotic features triggered by Erw-ASNase treatment on Kar-exposed U266 cells. Cleavage of PARP1, caspase-3, caspase-9, NRF2, IRF4, γ-H2A.X, and GAPDH (loading control) was assessed. One representative western blot of 3 is shown. (C) qPCR analysis for oxidative stress–induced genes in U266 cells treated with Erw-ASNase (0.5 U/mL), Kar (2.5 nM), or both. The dashed red line indicates the reference level of DMSO-treated cells. (D) MitoSOX staining on U266 cells upon Erw-ASNase treatment (0.5 U/mL), with or without Kar (5 nM) treatment, was evaluated by flow cytometry 28 hours posttreatment. All data are mean ± SD of 3 independent experiments. (E) U266 cells were incubated or not with NAC (5 mM) for 2 hours and then treated, in the presence or absence of NAC, with Erw-ASNase (0.5 U/mL), Kar (3 nM), or both. Mitochondrial superoxide levels were detected by immunofluorescence 24 hours later (left panels). Signals from MitoSOX staining were quantitated with ImageJ software and normalized to control (right panel). One representative experiment of 3 is shown. Magnification ×20. (F) Cell death of mitochondria-targeted catalase (mitoCat) and empty vector–overexpressing LP1 cells treated with the indicated stimuli (0.5 U/mL Erw-ASNase and 3 nM Kar). Cell death was assessed by flow cytometry using annexin V (AV) and propidium iodide (PI) double staining after 48 hours. The percentage of early apoptotic cells (AV+/PI−) are shown as white columns; late apoptotic cells (AV+/PI+) are shown as gray columns. One representative experiment of 3 is shown. *P < .05, **P < .01, ***P < .001, Student t test.

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