*4.4. EC50 Determination*

To quantitatively determine the effective potency in BV2 cells, the compound half maximal effective concentration or EC50 was determined by a dose-response curve. In brief, BV2 cells were plated in 96-well plates as described above and treated with increasing concentrations of compound Oxa12 (from 0.1 to 50 μM) and Nec-1 (from 0.01 to 50 μM) using DMSO as vehicle control. Following 24 h of incubation, cell viability was determined based on the measurement of MTS, as previously described.

## *4.5. RIP1 and RIP3 Kinase Activity Assays*

Oxa12 were tested at 5 μM for RIP1 kinase activity by a radiometric-binding assay using myelin basic protein as substrate (Eurofins, France) as before [39].
