Fig. 3.

Ferroptotic stress induces neurotoxic glial activation. a Conditioned medium from mixed glial cultures (i) treated with RSL3, erastin or LPS is toxic to cultured neurones (MTT reduction). Toxicity is alleviated by liproxstatin-1 (Lip1). Conditioned medium from identically treated isolated astrocyte (ii) or microglia (iii) cultures is not neurotoxic, whereas conditioned medium from isolated microglia is neurotoxic to neurones cultured with astrocytes (iv). Adding RSL3, erastin or LPS directly onto neurones is not toxic (v). Isolated microglia cultures treated with 1 nM RSL3. All other cultures treated with 100 nM RSL3. Error margins are S.E.M. P values indicate significant differences. Procedure for exposing neurones to glial conditioned medium is depicted in Supplementary Fig. 6a. Glial conditioned medium is concentrated using 30 kDa MWCO filters. Data points in a represent independent cultures. b Effect of RSL3 (200 nM) or erastin plus iron (as ferric ammonium citrate)-induced ferroptotic stress on expression of genes associated with activation of microglia and astrocytes in mixed glial cultures, and protection by Lip1 or deferiprone (DFN). c RSL3 (100–400 nM) induced expression of glial activation genes in mixed glial cultures (astrocytes & microglia) but not astrocytes alone. Individual heatmap values (b, c) represent mean (n = 3–6)