Anti active caspase 3

At the end of capsaicin and SMF treatments, cells were washed with PBS and fixed with 3.7% paraformaldehyde (PFA; Sigma) in PBS for 15 min at RT. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 15 min. Nonspecific protein binding was blocked with 1% BSA in PBS for 20 min at RT. After washing three times with PBS, the cells were incubated with anti-β-tubulin (1:1000 dilution; Abcam) and anti-active caspase-3 (1:800 dilution; Cell Signaling Technology) primary antibodies overnight at 4°C. The cells were then washed three times in PBS, and incubated in the Alexa Fluor 488-conjugated donkey anti-goat secondary antibody and the Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (1:500 dilution; Jackson ImmunoResearch Inc.) for 1 h at 37°C in the dark. The coverslips were mounted to slides in mounting medium with DAPI (Abcam). The mounted samples were examined by a Zeiss LSM 880 inverted laser scanning confocal microscope. Pictures of randomly selected areas were taken for each sample and representative micrographs are shown in the figures.

Mouse polyclonal antibodies against human BEX4 protein were generated in C57BL/6 mice. Briefly, purified GST-BEX4 protein was injected four times intraperitoneally. Rabbit polyclonal antibodies against C-terminal polypeptides 89–106 of human BEX4 protein were commercially generated (Youngin Frontier, Seoul, Korea). The other antibodies used in this study were as follows: anti-GFP, anti-PLK1, anti-CDK1, anti-aurora A, anti-cIAP-1, anti-cdc20 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-poly(ADP-ribose) polymerase-1 (PARP), anti-cleaved-caspase 9, cleaved-caspase 7, anti-active-caspase 3, anti-phospho-threonine (p-Thr) (Cell Signaling Technology, Danvers, MA, USA), anti-aurora B (BD Biosciences PharMingen, San Diego, CA, USA), anti-securin (PTTG1; Zymed, San Francisco, CA, USA), anti-Myc (Bethyl Laboratories, Montgomery, TX, USA), and Alexa Fluor (Invitrogen, Leek, The Netherlands). The following reagents were used: MG132, cycloheximide (CHX), dimethyl sulfoxide (DMSO) (AG Scientific, San Diego, CA, USA), nocodazole (Sigma-Aldrich), and PLK1 kinase inhibitor BI2536 (Axon Medchem, Groningen, Netherlands).

To determine whether cell death in the organ of Corti occurred via apoptosis, we performed immunohistochemistry for active caspase-3 and TUNEL assay. After fixation, specimens were permeabilized with 0.1% Triton X-100 in PBS (PBS-Tx) for 30 min at RT and blocked with 5% normal goat serum for 1 h at RT. The specimens were incubated with anti-active caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA, USA) diluted in blocking solution overnight. After three rinses with PBS, samples were labeled with Alexa Fluor^® 488-conjugated goat anti-rabbit immunoglobulin G (1:1000; Invitrogen, La Jolla, CA, USA) diluted in blocking solution for 1 h at RT. F-actin was labeled with Alexa Fluor^® 555-conjugated phalloidin in PBS-Tx for 1 h at RT.
To detect DNA fragmentation, a marker of apoptotic cell death, we used the TUNEL assay kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. Specimens were fixed with 4% paraformaldehyde in PBS for 15 min at RT and rinsed with PBS. And then, they were permeabilized with 0.1% PBS-Tx in 0.1% sodium citrate for 30 min at 37 °C and stained with TUNEL working solution for 30 min at 37 °C. F-actin was labeled with Alexa Fluor 555-conjugated phalloidin in PBS-Tx for 3 h at RT in the dark. Specimens were mounted on glass slides using Fluoromount and visualized using a Zeiss Axio Imager A2 fluorescence microscope.