Fig. 3.

Peroxynitrite- and Hsp90^NY-triggered PC12 cell death requires activation of p38 and JNK MAP kinases. A) Representative infrared western blot of an 8 h time course following p38 phosphorylation after intracellular release of Hsp90^NY. Blots were stained for phospho-p38 (P-p38, red), and total p38 and actin (green). The bottom graph shows the quantitation of the infrared fluorescence intensity of the P-p38 versus total p38 bands, expressed as the signal ratio. MW: molecular weight marker. The columns represent the mean ± SD (n = 3, individual values show as dots). Control (Ctr): Cells incubated with Chariot alone. *p < 0.001 vs control by one-way ANOVA followed by Holm-Sidak's multiple comparison test. B) Representative infrared western blot of a 6 h time course for JNK phosphorylation after intracellular delivery of Hsp90^NY. Blots were stained for phospho-JNK 54 (PJNK54), phospho-JNK 46 (PJNK 46), and GADPH (red), and total JNK 56 and JNK 46 (green). Bottom graph, quantitation of the infrared fluorescence signal ratio of PJNK 54 versus total JNK 54. No changes were detected in the phosphorylation of JNK 46 at any timepoint. The columns represent the mean ± SD (n = 3, individual values show as dots). Control (Ctr): Cells incubated with Chariot alone. *p < 0.05 vs control by Kruskal-Wallis test followed by Dunn's multiple comparison test. C) Viability of PC12 cell treated or not with peroxynitrite in the presence or absence of the p38 inhibitor SB203580 (1 μM; p38), the JNK inhibitor SP600125 (0.1 μM; JNK) and the selective caspase 3 inhibitor z-DVED-fmk (25 μM; Cas3) was evaluated 24 h after peroxynitrite-treatment. The values represent the mean ± SD (n = 3 with 8 replicates). *p < 0.001 vs control, ** vs ONOO⁻ by one-way ANOVA test followed by Holm-Sidak's multiple comparison test. D) PC12 cell viability was assessed 24 h after the intracellular release of Hsp90^NY in the presence or absence of the p38 inhibitor SB203580 (1 μM; p38), the JNK inhibitor SP600125 (0.1 μM; JNK), and the selective caspase 3 inhibitor z-DVED-fmk (25 μM; Cas3). In all cases, the values represent the mean ± SD (n = 3 with 3–8 replicates). *p < 0.001 vs control, ** vs Hsp90^NY by one-way ANOVA followed by Holm-Sidak's multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)