Tigar

Approximately 4 million cells were harvested with RIPA lysis buffer with phosphatase inhibitor (Thermo Scientific, no. A32957) and HALT protease inhibitor (Thermo Scientific, no. 78425). Proteins of interest were probed with the following antibodies in TBS-T buffer with 5% BSA. Primary antibodies: G6PD (abcam, no. ab993), TIGAR (abcam, no. ab37910), Beta-actin (Cell Signaling, no. 3700 S); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, no. 925-32211), Goat-anti-mouse 680 (LI-COR, no. 925-68070).

Cells were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Mixture (Thermo Scientific). Lysates were spun at 16,000×g at 4 °C for 30 min and normalized for protein concentration. Western blotting was performed as follows: total tumor lysates were separated by SDS/PAGE and electrotransferred to nitrocellulose membrane (Invitrogen). Membranes were blocked in PBS and 0.1% (vol/vol) Tween-20 (PBS-T) and 4% (wt/vol) nonfat dry milk (Bio-Rad) for 1 h on a shaker at room temperature. Primary antibodies were added to the blocking solution at 1:500 (TIGAR; Abcam, 37910), 1:500 (GSH; Abcam, 19534), 1:500 (PFKFB3; Abcam, 96699), and 1:1000 (Actin; Abcam, 3280) dilutions and incubated overnight and a rocker at 4 °C. Immunoblottings were washed three times, 5 min each with PBS-T, and secondary antibody was added at 1:10,000 dilution into PBS-T milk for 1 h on a shaker at room temperature. After several washes, enhanced chemiluminescence (ECL) reactions were performed according to the manufacturer’s recommendations (SuperSignal West Dura Extended Duration Substrate; Thermo Scientific).

Protein lysates were prepared in RIPA buffer (Thermo Fisher Scientific, Cat#89900), resolved via SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (Millipore, Cat#ISEQ00010). The following primary antibodies were used: TIGAR (Abcam, Cat#ab62533), Caspase-3 (CST, Cat#9662), β-actin (Abcam, Cat#ab8226). After incubation with HRP-conjugated secondary antibodies, bands were detected and visualized by FluorChem M (ProteinSimple, San Jose, CA, USA).

After culture, cochlea explants or HEI-OC1 cells on coverslips were fixed with 4% paraformaldehyde and blocked for 1 h at room temperature with PBT-1 (5% donkey serum, 0.1% Triton X-100, 1% bovine serum albumin and 0.02% sodium azide in PBS). The samples were then incubated overnight at 4℃ with primary antibodies diluted in blocking solution against cleaved-Caspase 3 (1:500 dilution; Cell Signaling Technology) and TIGAR (1:800 dilution; Abcam). The next day, the samples were incubated with secondary fluorescent antibodies (1:1000 dilution, Invitrogen) along with DAPI (1:1000 dilution, Sigma-Aldrich) in 0.1% Triton X-100 and 1% bovine serum albumin in PBS at room temperature for 1 h. The coverslips were mounted and imaged using a confocal laser scanning microscope (Leica SP8; Leica, Germany).
For the staining of phalloidin, after fixation, the basilar membranes on coverslips were incubated with iFluor™ 488 phalloidin (1:1000 dilution; Yesen) in PBS along with DAPI (1:1000 dilution) for 30 min in the dark. The coverslips were then mounted and confocal microscopy images were acquired.