Figure 2. KEAP1 binds PGAM5, which is required to induce ROS-dependent cell death.

(a) Identification of KEAP1 binding partners. SII-Affinty purification (AP) of SII-HA-tagged KEAP1 and THYN1 as control (ctrl) followed by LC-MS/MS analysis. Volcano plots show the average degree of enrichment of label-free quantitation (LFQ) values by KEAP1 over control (x-axis) and transformed p-values (two-tailed t-test; y‐axis) for each identified protein. The hyperbolic curve separates significantly enriched proteins (FDR < 0.00001, S0=100) background. Red: KEAP1, brown: known KEAP1 interactors (based on Biogrid), green dot: protein associated with cell death. Four independent APs were performed for each bait. (b) Immunoblot of precipitates after AP with SII-HA-KEAP1 or SII-HA-THYN1 (ctrl). (c) SII-HA-KEAP1 expressing HeLa Flp-In cells were treated with the indicated amounts of H₂O₂ for 8h followed by SII-AP and Immunoblotting. (d) Flow cytometry analysis for viability of HeLa cells transfected with the indicated siRNAs for 48h, followed by treatment with 0.5 mM of H₂O₂ for the indicated time. (e) HeLa cells were treated with the indicated siRNAs for 48h and stimulated with H₂O₂ for 20h. The graph shows mean resazurin conversion activity ± S.D. of six measurements. Knockdown efficiency was confirmed by Immunoblotting against indicated proteins 48h after siRNA transfection (right panel). (f) as (e) but stimulation with H₂O₂, 20 µM CCCP, 20 µM Sorafenib (Sora), 20 µM Z-VAD + 20 µM TNF and 1 µM STS,, respectively. (g) as (e) but MEFs with indicated genotype were stimulated as indicated. (h) Light microscopic images of Pgam5^+/+ and Pgam5^−/− MEFs after 20h treatment with 1 mM H₂O₂. Scalebar: 100 µM. (e)-(g) ** p-value < 0.001, * p-value < 0.05, ns: non-significant, 2way ANOVA. One representative experiment of three (b)-(f) (h), four (g) or six (e) is shown.