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. 2021 Apr 23;43:101989. doi: 10.1016/j.redox.2021.101989

Fig. 3.

Fig. 3

NO contributes to PAL-induced ferroptosis of MM cells via lysosomal localization with peroxynitrite formation. (A) Contribution of . NO to ferroptosis evaluated by cell viability. MM cells were treated with PAL for 15 min, followed by incubation for 4–24 h with or without caboxy-PTIO, 1400 W or Fer-1 (H290, sarcomatoid MM; 8A, epithelioid MM). (B) Contribution of . NO to ferroptosis evaluated by lipid peroxidation. MM cells were treated with PAL for 15 min, followed by incubation for 0.5–8 h with or without Fer-1 or the combination of caboxy-PTIO and 1400 W, and then incubated with BODIPY™ 581/591 C11. Lipid peroxidation was evaluated by FACS analysis. (C) Lysosomal . NO after PAL exposure. After treatment with PAL (15 min) and an incubation of 4 h with or without Fer-1, H290 cells were stained with either DAF-FM DA (green) or LysoTracker Red DND-99 (red) and Hoechst 33342. The white punctate staining indicates electronic merge (Merge) of DAF-FM DA and LysoTracker by confocal microscopy (bar = 20 μm). (D) Induction of lysosomal lipid peroxidation after PAL exposure. After treatment with PAL (15 min) and an incubation of 4 h with or without Fer-1, H290 cells were stained with either BODIPY™ 581/591 C11 or LysoTracker Red DND-99 (red). The white punctate staining indicates electronic merge (Merge) of BODIPY™ 581/591 C11 and LysoTracker by confocal microscopy (bar = 20 μm). (E) Peroxynitrite production after PAL exposure. H290 cells were treated with PAL for 15 min, followed by incubation with or without Fer-1 or the combination of caboxy-PTIO and 1400 W for an additional 0.5–8 h with Peroxynitrite Sensor Green working solution. (F) Erastin-induced ferroptosis is not NO-dependent. MM cells were treated with erastin in the presence or the absence of caboxy-PTIO, 1400 W or Fer-1 for an additional 4–24 h. Cells treated with PAL for 15 min were taken as positive control. Representative data are shown from 3 experiments and the analysis is shown as means ± SEM (N = 3), *P < 0.05, **P < 0.01, ***P < 0.001 vs each RL unless indicated by bar. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)