Research Paper Volume 13, Issue 8 pp 11010—11025

Figure 5. Obacunone-induced RPE cytoprotection against UVR is abolished with Nrf2 silencing or knockout. Stable ARPE-19 cells with Nrf2 shRNA (“sh-Nrf2”) or the CRISPR/Cas9-Nrf2-KO construct (“ko-Nrf2”), as well as the control cells (“sh-c+Cas9-C”), were treated with obacunone (25 μM), cells were further cultured for 6h, expression of listed genes was shown (A, B), with ARE activity tested (C). Alternatively, cells were pretreated with obacunone (25 μM) for 1h, followed by UVR and cultured for another 48h, cell viability and death were tested by CCK-8 (D) and medium LDH release (E) assays, respectively. The primary murine RPE cells were transfected with Nrf2 siRNA (“si-Nrf2”, 500 nM) or the scramble control siRNA (“si-C”, 500 nM), after 48h cells were treated with obacunone (25 μM) for another 6h, expression of listed proteins was shown (F). The murine RPE cells were pre-treated with obacunone (25 μM) for 1h, followed by UVR for another 48h, cell viability (G) and death (H) were tested. Expression of listed proteins was quantified, normalized to the loading control, and expressed as mean ± SD (n=5) (B, F). Data were presented as mean ± SD (n=5). ^# p < 0.05 vs. “sh-c+Cas9-C” cells (B–E). ^# p < 0.05 vs. “si-C” cells (G, H). Experiments were repeated three times, with similar results obtained.