Fig. 4.

Nrf2 activation mediates 4-octyl itaconate-induced neuronal cell protection against H₂O₂. SH-SY5Y cells (a-e) or the primary murine neurons (i-k), with the applied Nrf2 shRNA or the scramble control shRNA (“shC”), were either untreated or treated with 4-octyl itaconate (OI), mRNA and protein expression of listed genes were shown (a-c, and i); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H₂O₂ (300 μM) for indicated time, cell viability (CCK-8 OD, d), cell death (LDH release, j) and apoptosis (TUNEL ratio increase, e, and k) were tested. Stable SH-SY5Y cells, with the CRISPR/Cas9-Nrf2 KO construct (“Nrf2-KO”) or the CRISPR/Cas9 control construct (“Cas9-c”), were treated with 4-octyl itaconate (OI), listed proteins were shown (f); Cells were pretreated for 30 min with 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 (c, f and i). “shNrf2 (m)” stands for murine Nrf2 shRNA (I-K). Bars stand for mean ± standard deviation (S.D., n = 5). ^# P < 0.05 vs. “shC” cells (a, b, d and e). ^# P < 0.05 (g, h, j and k)