Research Paper Volume 13, Issue 5 pp 6712—6723

Figure 2. CPI-1189 inhibits OGDR-induced oxidative injury in neuronal cells. Stable SH-SY5Y cells with CRISPR/Cas9-p38α-KO-GFP (ko-p38α cells) were pretreated with or without CPI-1189 (100 nM, 1h pretreatment), control cells were transduced with the empty vector (“Cas9-C”), cells were subjected to OGDR procedure and cultured for applied time periods; Expression of listed proteins was shown (A); Cell viability and death were tested by CCK-8 (B) and Trypan blue staining (C) assays, respectively. SH-SY5Y cells (D–H) or primary murine cortical neurons (K–N) were pretreated for 1h with CPI-1189 (100 nM), followed by OGDR or hydrogen peroxide (H₂O₂, 300 μM) stimulation, cells were then cultured for applied time periods, cellular ROS contents (CellROX dye intensity, D, K), lipid peroxidation (by recording TBAR activity, E), and GSH/GSSG ratio (F–L) were tested. For cells with H₂O₂ stimulation, cell viability and death were tested by CCK-8 (G–M) and Trypan blue staining (H–N) assays, respectively. The primary murine cortical neurons were pretreated for 1h with SB203580 (5 μM) or plus CPI-1189 (100 nM), followed by OGDR stimulation and cells were then cultured for 48h; Cell viability and death were tested by CCK-8 (I) and Trypan blue staining (J) assays, respectively. * P < 0.05 vs. “Mock” cells. ^# P < 0.05 vs. cells with OGDR stimulation/H₂O₂ treatment but “DMSO (0.1%)” pretreatment. ** P < 0.05 (B, C, I, J). Quantified values were mean ± standard deviation (SD, n=5). Experiments were repeated three times, with similar results obtained. Scale bar= 100 μm (D).