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. Author manuscript; available in PMC: 2014 May 1.
Published in final edited form as: Cell Rep. 2013 Nov 21;5(4):878–885. doi: 10.1016/j.celrep.2013.10.034

Figure 4. Elimination of mitochondria prevents necroptosis-associated ROS production, but does not alter RIPK3-dependent cell death.

Figure 4

A. NIH-3T3 cells expressing acRIPK3 and Parkin were treated with CCCP to induce mitochondrial elimination, then treated with TNF+zVAD for 2 hours in the presence of the ROS scavangers butyrated hydroxyanisole (BHA) or N-acetylcysteine (NAC). ROS production was measured using the dye DCF. MFI=Mean Fluorescent Intensity. Results shown are the mean ± S.D. of n≥3 independent experiments. *, P<0.001. Black dotted line corresponds to the basal level of ROS in the Control at t=0. B. NIH-3T3 cells treated like in A for 4 hours, were stained with PI and cell death was determined by flow cytometry (mean ± S.D. of n≥3 independent experiments). *, P<0.05, **, P=0.0518 (t test). C. Mitochondria were depleted as in A, and RIPK3 dimerization was triggered in the presence of BHA or NAC. 2 hours later cell death was quantified as in B. (mean ± S.D. of n≥3 independent experiments) D. Mitochondria depletion was triggered in NIH-3T3 cells as in A, and cells were then treated with TNF+zVAD and BHA as indicated. Cell death was measured by Sytox Green uptake over time in an IncuCyte imager. Data show one representative experiment of three independent experiments. See also Figure S3.