Fig. 5.

Keap1 knockout or Cys151S mutation abolishes 4-octyl itaconate-induced neuroprotection against H₂O₂. Stable SH-SY5Y cells with the lentiviral CRISPR/Cas9 Keap1 vector (“Keap1-KO”) or the CRISPR/Cas9 control vector (“Cas9-c”) were treated with/without 4-octyl itaconate (“+OI”, 25 μM, for 12 h), mRNA and protein expression of listed genes were shown (a and b). Cells were pretreated with/without OI (“+OI”, 25 μM), followed by stimulation of H₂O₂ (300 μM) for indicated time, cell viability (c) and apoptosis (d and e) were tested by the listed assays. Stable SH-SY5Y cells with the Cys151S mutant Keap1 [“Keap1 (c151s)”] or the empty vector (“Vector”) were treated with 4-octyl itaconate (“OI”, 25 μM) for 12 h, expression of listed proteins were shown (f). Cells were pretreated with/without OI (“+OI”, 25 μM), followed by stimulation of H₂O₂ (300 μM) for indicated time, cell viability (g), and apoptosis (h) were tested. Expression of listed proteins were quantified and normalized to the loading control (a and f). Bars stand for mean ± standard deviation (S.D., n = 5). * P < 0.05 vs. “Cas9-c” cells (b). * P < 0.05 vs. “Ctrl” cells (c-e). ^# P < 0.05 vs. H₂O₂ treatment of “Cas9-c” cells (c-e). ^# P < 0.05 (g and h)