duced destruction of the hair cell arrangement. In addition, we used the NO scavenger C-PTIO to confirm that NO mediated the effects of SNAP. As shown in Fig. 1E, CPTIO inhibited SNAP-induced destruction of the hair cell Regulatory effects of EGCG on NO-induced apoptosisrelated gene expression in HEI-CO1 cells Western blot analysis was performed to assess the effects of SNAP on the release of cyt c into the cytosol. SNAP induced the release of cyt c into the cytosol, and EGCG inhibited this process. The relative quantity of cyt c was determined using an image analyzer. As shown in Fig. 5C, EGCG also inhibited the reduction in Bcl-2 levels induced by SNAP. Relative Bcl-2 expression is shown in Fig. 5D. Next, we performed western blotting and a caspase-3 activity assay to determine whether NOinduced apoptosis was associated with the regulation of caspase-3 activity. SNAP increased the expression of caspase-3, while EGCG effectively inhibited this increase. EGCG also attenuated the SNAP-induced increase in caspase-3 activity. EGCG Protects Auditory Cells MedChemExpress RS-1 against NO Damage Protective effects of EGCG on NO-induced NF-kB signaling in HEI-CO1 cells To determine the association of NO-induced apoptosis with the NF-kB pathway, we silenced endogenous NF-kB using specific siRNA. The siRNA effectively inhibited NF-kB expression in the nucleus relative to control cultures transfected with scrambled siRNA. As shown in Fig. 6B, knockdown of NF-kB was effective at inhibiting SNAP-induced caspase-3 activation. The siRNA transfections resulted in 52% and 48% knockdown of NF-kB and caspase-3, respectively. Based on these findings, we investigated the relationship between the protective mechanisms of EGCG and regulation of the NF-kB pathway. Our results revealed that SNAP induced the degradation of IkB-a in the cytosol and translocation of NF-kB into the nucleus; EGCG suppressed these SNAP-induced phenomena. Next, we performed a luciferase assay to investigate the effects of EGCG on NF-kB promoter activity. As shown in Fig. 6E, SNAP treatment enhanced NF-kB promoter activity, while EGCG pretreatment inhibited this SNAP-induced increase in NF-kB promoter activity. Immunofluorescent staining of NF-kB and nuclei revealed that SNAP treatment caused translocation 
of NF-kB into the nucleus, while pretreatment with EGCG inhibited this phenomenon. Protective effects of EGCG on NO-induced NF-kB activation in organ of Corti explants Next, we investigated the regulatory effects of SNAP on NF-kB activation ex vivo. As shown in Fig. 7, treatment with SNAP induced NF-kB activation in the organ of Corti, and EGCG inhibited SNAP-induced NF-kB activation. Protective effect of EGCG on NO-induced caspase-1 activation in HEI-CO1 cells and organ of Corti explants We investigated whether NO-mediated ototoxicity occurred via the production of IL-1b and activation of caspase-1. As shown in Fig. 8A and B, SNAP induced IL-1b production and increased the levels of caspase-1 in HEI-CO1 cells, while EGCG inhibited these effects. To confirm the effects of EGCG on caspase-1 activation ex vivo, we performed a caspase-1 activity assay in organ of Corti explants. The results demonstrated that SNAP induced caspase-1 activation, and this effect was again inhibited by EGCG. Discussion We have shown, for the first time, that EGCG is effective in preventing the destruction of hair cell arrays and apoptosis both in vitro and ex vivo. EGCG is also effective in counteracting ototoxicity by suppress
