Inhibition of JNK activation with simultaneous stimulation of protein synthesis results in protection from cisplatin ototoxicity. (A) Cisplatin exposure (100 μM) results in significant loss of outer hair cells (second row). Co-incubation of cisplatin with either insulin or sorafenib results in a small, but significant protection of outer hair cells (3rd, 4th rows). Combined incubation with cisplatin, insulin and sorafenib provided strong protection of cochlear hair cells (5th row). This protection, however, did not persist when organs were cultured for another 24 h in normal growth medium (7th row). Addition of rapamycin, a known inhibitor of the mTOR pathway, did not reverse the protection seen with sorafenib and insulin (6th row). (B) Quantification of outer hair cell numbers demonstrating the protective effect of insulin (Ins), sorafenib (Sora) and the combination of the two after 100 μM cisplatin (Cis) exposure. P-values for analysis of variance (ANOVA) with post hoc test (Tukey): Con to Cis: < 0.0001, Cis to Cis+Ins and Cis+Sora: < 0.001, Cis to Cis+Ins+Sora: < 0.0001. (C) Same as (B), but after 200 μM cisplatin exposure. P-values for ANOVA with post hoc test (Tukey): Con to Cis: < 0.0001, Cis to Cis+Ins and Cis+Sora: < 0.001, Cis to Cis+Ins+Sora: < 0.0001. (D) Quantification of cleaved Caspase-3 (Casp-3)-positive cells, demonstrating the protective effect of insulin (Ins), sorafenib (Sora) and the combination of the two after 100 μM cisplatin (Cis) exposure. P-values for ANOVA with post hoc test (Tukey): Con to Cis: < 0.001, Cis to Cis+Ins and Cis+Sora: < 0.001, Cis to Cis+Ins+Sora: < 0.0001. (E) Same as (D), but after 200 μM cisplatin exposure. P-values for ANOVA with post hoc test (Tukey): Con to Cis: < 0.0001, Cis to Cis+Ins and Cis+Sora: < 0.0001, Cis to Cis+Ins+Sora: < 0.0001. Asterisks in plots indicate p-values (***< 0.0001, **< 0.001). To reduce unnecessary complexity in the plots, only a subset of the p-values of the pair-wise post hoc tests are displayed. Inner hair cells were not affected in all experimental groups (not included in quantification). Scale bar 20 μm.