Fig. 1.

Nitration of Hsp90 at Y56 precedes peroxynitrite triggered-PC12 cell death. PC12 cells were incubated with 0.5 mM peroxynitrite (ONOO⁻), or the products of peroxynitrite decomposition (ROA), as described in Materials and Methods. A) Representative infrared western blot showing nitroTyr staining (green) after treatment of PC12 cells with peroxynitrite. Tubulin (Tub) infrared signal was used as a loading control (red). B) Representative infrared western blot showing nitration of Hsp90 at Y56 (Hsp90^NY56 in red, upper panel). In green, infrared signal for total Hsp90 (middle panel), Tubulin in red (lower panel). STD: Peroxynitrite-treated recombinant human Hsp90; MW: molecular weight marker. C) Representative image of PC12 cells 6 h post ROA or ONOO⁻ treatment stained for Hsp90^NY56 (green) and nuclei (DAPI, blue). D) Representative infrared western blot stained for Hsp90 specifically nitrated at Tyr 33 (Hsp90^NY33 in red), tubulin (red), and for total Hsp90 (green). E) Viability of PC12 cells incubated in the absence (control) or presence of ONOO⁻, or ROA. F) Representative images of live (green) and death (red) PC12 cells 24 h post-treatment with ONOO⁻ or ROA. G-H) PC12 cell viability was measured 24 h after the intracellular delivery of Hsp90 or Hsp90 in which all 5 residues prone to nitration were replaced by phenylalanine (Hsp90^5F) (G), or Hsp90 with nitroY at either position 33 (Hsp90^33NY) or 56 (Hsp90^56NY), or Hsp90 with Y56 and phenylalanine in the other 4 residues prone to nitration (Hsp90^4F56Y) (H), treated or not with ONOO⁻, and in the absence (Control) or presence of the permeant agent Chariot. In all cases, the values represent the mean ± SD (n = 3–5 with 3–8 replicates). *p < 0.01 vs control by Kruskal-Wallis test followed by Dunn'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.)