Supplementary Information
Supplementary Method
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed similarly as in Bai et al . [1]. In HEI-OC1 cells, chromatin-bound proteins were fixed on DNA by formaldehyde. Nuclei were prepared and chromatin was sonicated in order to split DNA into shorter, ~500 bp fragments. PARP-2bound chromatin fragments were collected by immunoprecipitation using anti-PARP-2
(Santa Cruz, Santa Cruz, CA, USA) antibody. Formaldehyde crosslinking was reversed by heating and then DNA fragments were purified and assayed in quantitative RT-PCR using primer specific for the -1 ~ -91 portion of the SIRT1 promoter.
Supplementary Figure Legends
Figure 1. Effect of

-Lap on cisplatin-induced toxicity in HEI-OC1 auditory cells. (A) HEI-OC1 cells were pretreated with the indicated doses of

-Lap for 30 min, after which they were further maintained in 20

M cisplatin for 24 h. (B) Cells were transfected with 100 nM control or NQO1-specific siRNAs, and then incubated for 24 h. The cells were further treated with

-
Lap (2

M) and cisplatin (20

M) for 24 h. The cell viability was then measured by MTT assay. * indicates p < 0.05 by one-way ANOVA compared with cisplatin treated cells. N.S.: not significant.
Figure 2. Effect of

-Lap on intracellular NAD + and NADH content in cisplatin-treated HEI-
OC1 cells. Cells were treated with various doses of

-Lap for 24 h in the presence or absence of 20

M cisplatin. (A) NAD + content and (B) NADH content were measured by using the NAD + /NADH assay kit. *, # indicate p < 0.05 by one-way ANOVA compared with the control (*) and cisplatin-only group (#).

Figure 3. Effect of

-Lap on cellular ATP levels.

-Lap (40 mg/kg body weight) was administered orally once a day for 4 consecutive days. Cisplatin (16 mg/kg body weight) was injected once at 12 h after the first

-Lap administration. Cochlear tissue was isolated at indicated periods (A) or 24 h (B). HEI-OC1 cells were treated with 20

M cisplatin for the indicated times (C) or 24 h (D). Intracellular ATP levels were measured using ATP assay kit in the cochlear tissue (A, B) and HEI-OC1 cells (C, D) on a luminometer. *, # indicate p <
0.05 by one-way ANOVA compared with the control (*) and cisplatin only (#) group (n = 5).
Figure 4. The expression and activity of SIRT1 were decreased by cisplatin in HEI-OC1 auditory cells. HEI-OC1 cells were treated with 20

M cisplatin for the specified periods.
Western blotting was performed using anti-SIRT1 antibody, and signal intensity was quantified using the ImageJ program (A). After treatment with cisplatin (20

M) for 18 h, immunocytochemistry was performed with DAPI and anti-SIRT1 antibody, and then cells were visualized under fluorescent microscope (B). SIRT1 enzyme activity was measured using the SIRT1 assay kit (C). Cells were pretreated with the indicated doses of

-Lap for 30 min, after which they were further maintained in 20

M cisplatin for 24 h. Level of SIRT1 protein was analyzed by western blotting (D). SIRT1 activity was measured using SIRT1 assay kit (E). *, # indicate p < 0.05 by one-way ANOVA compared with the control (*) and cisplatin only (#) group (n = 3).
Figure 5.

-Lap attenuates cisplatin-induced increase in Bax and p21 expression. (A) HEI-
OC1 cells were treated with 20

M cisplatin for the indicated periods. (B) Cells were treated with various doses of

-Lap for 24 h in the presence or absence of 20

M cisplatin. Levels of
Bax and p21 protein were analyzed by western blotting, and signal intensity was quantified using the ImageJ program. *,# indicate p < 0.05 by one-way ANOVA compared with the control (*) and cisplatin only (#) group (n = 3).

Figure 6.

-Lap inhibits cisplatin-induced production of pro-inflammatory cytokines. (A)
Expression of TNF-

was analyzed by IHC in the cochlear tissue. Control, PBS-treated group; Cisplatin, 16 mg/kg cisplatin only group; Cisplatin+

-Lap, cisplatin and 40 mg/kg

-
Lap combined group;

-Lap, 40 mg/kg

-Lap only group. (B-D) mRNA expression levels of
TNF-

, IL-1

, and IL-6 were analyzed by quantitative RT-PCR in the cochlear tissue. *, # indicate p < 0.05 by one-way ANOVA compared with the control (*) and cisplatin only (#) group, (n = 5). SL: spiral ligament; SV: stria vascularis; SGN: spiral ganglion neuron; SLim: spiral limbus; OC: organ of Corti.
Figure 7. PARP-2 did not bind directly to SIRT1 promoter in cisplatin-treated HEI-OC1 cells.
HEI-OC1 cells were treated with 20

M cisplatin for the specified periods. Western blotting was performed using anti-PARP-2 antibody (A). Chromatin immunoprecipitation was performed using anti-PARP-2 antibody, and analyzed using primers specific to the SIRT-1 promoter regions (B).
Supplementary Reference
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