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. 2021 Feb 8;12(2):159. doi: 10.1038/s41419-021-03454-9

Fig. 3. Production of ROS is required for tetraarsenic hexoxide-induced pyroptosis in TNBC cells.

Fig. 3

A Representative transmission electron microscopy images of 4T1 cells treated with 5 μM tetraarsenic hexoxide for 24 h. Scale bar, 200 nm. B Representative immunoblot analysis showing cytochrome c expression in mitochondria fractions. Cells were treated with 5 μM tetraarsenic hexoxide for 24 h and then mitochondria were fractionated. HSP60 was used as an internal control of mitochondria fractions. C–E Quantification of TMRE fluorescence intensity (C) and confocal images and quantification of JC-1 dye (D, E) showing mitochondrial membrane potential in TNBC cells treated with 5 μM tetraarsenic hexoxide for 24 h. F Flow cytometry analysis showing cellular ROS levels in TNBC cells. Cells were treated with 5 μM tetraarsenic hexoxide for 24 h and then stained with DCFDA. G Cells were pretreated with or without 5 mM NAC for 2 h before treatment of 5 μM tetraarsenic hexoxide for 24 h, and then analyzed using a flow cytometer. H–K Representative immunoblot analysis (H), Phase-contrast images (I), LDH release (J), and flow cytometry analysis (K) showing ROS-mediated pyroptotic characteristics induced by tetraarsenic hexoxide upon pretreatment of NAC in TNBC cells. Cells were pretreated with or without 5 mM NAC for 2 h before treatment of 5 μM tetraarsenic hexoxide for 24 h. Pyroptotic cell morphology was indicated by white arrows. Original magnification, ×200. Scale bar, 50 μm (I). The data represent the mean ± S.D. of three independent experiments. ** P < 0.01, ***P < 0.001 using unpaired two-tailed Student’s t-tests (C, E and J). The data represent the mean ± S.D. of three independent experiments. FL full length, N N-terminus.