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Sephin1 reduces mitochondrial ROS without modulating calcium flux or the phosphorylation of eIF2α in primary rat motor neurons against glutamate intoxication. (A) Calcium influx level in primary WT rat motor neurons treated with DMSO, Sephin1 or riluzole measured 5 min after the glutamate intoxication. (B) Representative pictures obtained by WES automated WB apparatus of the eIF2α phosphorylated and eIF2α immunoblots in primary WT rat motor neurons intoxicated with glutamate and treated with DMSO or Sephin1 50 nM. (C) eIF2α phosphorylation level in primary WT rat motor neurons intoxicated with glutamate and treated with DMSO or Sephin1 50 nM 24 h after glutamate intoxication. (D) Representative pictures obtained by WES automated WB apparatus of the eIF2α phosphorylated and eIF2α immunoblots in primary SOD1G93A rat motor neurons intoxicated with glutamate and treated with DMSO or Sephin1 50 nM. (E) eIF2α phosphorylation level in primary SOD1G93A rat motor neurons intoxicated with glutamate and treated with DMSO or Sephin1 24 h after glutamate intoxication. (F) Representative pictures of mitoSOX staining (top panel) in primary SOD1G93A rat motor neurons enriched culture intoxicated with glutamate and treated with DMSO or Sephin1 500 nM, 4 h after glutamate intoxication. Representative images of mitoSOX staining overlapped with MAP-2 staining and Hoechst (bottom panel) in primary SOD1G93A rat motor neurons enriched culture intoxicated with glutamate and treated with DMSO or Sephin1 500 nM, 4 h after glutamate intoxication. White arrow: mitoSOX staining in MAP-2 labeled motor neurons. (G) Mitochondrial ROS level in primary SOD1G93A rat motor neurons intoxicated with glutamate and treated with DMSO or Sephin1. #P < 0.05, ##P < 0.01 versus 0 nM Sephin1 Mann-Whitney test; **P < 0.01 versus 0 nM Sephin1 Kruskal-Wallis test followed by Dunn’s multiple comparison test; mean ± SEM n = 4–6 replicates per condition. Source data are available for this figure.
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