Figure 8.

Unbiased phosphoproteomics of U0126 treatment in ferroptotic cell death. A, Primary neurons were treated with 10 μm U0126 for 7 or 5 h in erastin-treated or hemin-treated cells. A total of 34 phosphopeptides from 34 proteins were significantly altered in hemin+U0126-treated versus hemin-treated cells and 61 phosphopeptides from 58 proteins in erastin+U0126-treated versus erastin-treated cells, with no overlap between both groups (p < 0.001, for full list of phosphopeptides, refer to Extended Data Fig. 7-1). B, Kinases enriched for proteins with phosphopeptides that were downregulated by U0126 compared with either erastin or hemin treatment alone. The top 10 kinases are ordered by the mean rank of enrichment over 11 gene set libraries (see also Extended Data Fig. 7-2). For top 10 ranked kinases upregulated in erastin+U0126-treated cells, see Extended Data Figure 8-1. C, Analysis of phosphopeptides that were significantly changed in hemin versus control treatment, that also had significantly different levels between the hemin-treated and erastin-treated cells, and where U0126 treatment led to changes in the opposite direction of the hemin treatment identified 247 phosphopeptides with significantly altered levels (p < 0.05, see also Extended Data Fig. 8-2). Dashed lines indicate a p value threshold of 0.05. D, Motif analysis shows that Rxxs (CAMK2, Akt, PKA, DMPK1/2), (s/t)xx(E/D) (CK2), and sP (MAPK and other kinases) were enriched versus their abundance in the proteome. sxxD/E was overrepresented (ratio 2.06 or 2.05, respectively, see also Extended Data Fig. 8-3). E, F, Network analysis of the proteins revealed some GO cellular processes (E) and functions (F) that provide the basis for a future search for the target of U0126 in hemin-induced ferroptosis.