Research Paper Volume 12, Issue 13 pp 13388—13399

Figure 5. In Keap1-KO or PGK1-KO SH-SY5Y cells CBR-470-1 fails to offer further cytoprotection against MPP⁺. Expression of listed mRNAs and proteins in empty vector (“Vec”)-expressing SH-SY5Y cells or stable SH-SY5Y cells with a lenti-CRISPR/Cas9-Keap1 KO construct (“ko-Peak1”), treated with or without CBR-470-1 (“CBR”, 10 μM, for 4-8h), was tested by qPCR (A, D) and Western blotting (B, E) assays, with the relative ARE luciferase activity tested as well (C); Alternatively, cells were treated with MPP⁺ (3 mM) for 48h, cell viability and death were tested by CCK-8 (F) and medium LDH release (G) assays, respectively. The ko-PGK1 cells were pre-treated for 2h with CBR-470-1 (“+CBR”, 10 μM), followed by MPP⁺ (3 mM) stimulation for 48h, with cell viability (H) and death (I) tested similarly. Expression of listed proteins was quantified and normalized to the loading control (B, E). Bars stand for mean ± standard deviation (SD, n=5). * P< 0.05 vs. “Vec” cells (A, C, D). * P< 0.05 vs. “Vec” cells with Ctrl treatment (F, G). ^#P< 0.05 vs. MPP⁺-treated “Vec” cells (F, G). * P< 0.05 vs. “Ctrl” treatment (H, I). Experiments in this figure were repeated four times, with the similar results obtained.