Previously we described the development of 7-Cl-O-Nec-1 (Nec-1) as a potent and selective inhibitor of RIP1 kinase and necroptosis (Fig. S1A) [23], [24]. Recently, its selectivity has been further validated against a panel of more than 400 human kinases [15]. This inhibitor efficiently blocked growth factor/zVAD.fmk-induced necroptosis under serum free conditions in L929 cells and both zVAD.fmk and TNFα-induced necroptosis under full serum conditions (Fig. 1B, S1B). To further validate the role of RIP1, we used an inactive analog, 7-Cl-O-Nec-1i (Nec-1i) (Fig. S1A), which contains an extra N-methyl group that leads to almost complete loss of RIP1 kinase inhibitory activity in vitro [23]. Nec-1i was unable to protect L929 cell death under serum condtions treated with zVAD.fmk or TNFα (Fig. S1B) or serum free conditions treated with bFGF/zVAD.fmk (Fig. S1C). This confirms that RIP1 kinase is responsible for necroptosis in L929 cells under both serum and serum free conditions.