SPATA2 is required for TNFα-induced necroptosis in BMDMs and MEFs. (A,B) Primary Spata2+/+ or Spata2−/− BMDMs were treated with DMSO, 1 µM IDN-6556, or 20 ng/mL mouse TNFα for 16 h. Cell viability and cell death were evaluated using CellTiterGlo and ToxiLight assay, respectively. (C) Primary Spata2+/+ or Spata2−/− MEFs were treated with DMSO, 1 µM IDN-6556, 20 µM zVAD.fmk, or 20 ng/mL human TNFα for 16 h. Cell death was measured by ToxiLight assay. (D) Immortalized Spata2+/+ or Spata2−/− MEFs were pretreated with Nec-1s for 30 min and treated with 20 µM zVAD.fmk and 20 ng/mL human TNFα for the indicated periods of time. Cell viability was measured by CellTiterGlo assay. (E) Immortalized Spata2−/− MEFs and Spata2+ MEFs were pretreated with or without 100 nM SM-164 for 30 min and then treated with 10 ng/mL human TNFα and 50 µM zVAD.fmk for 24 h. Cell viability was measured by CellTiterGlo assay. (Right) Western blotting was used to determine the levels of Spata2. Error bars represent standard error of the mean (SEM) from three technical replicates.