Fig. 5.

Activation of PTEN mediates decreased Akt phosphorylation and PC12 cell death triggered by peroxynitrite, nitrated Hsp90, and activation of P2X7R. A) Representative infrared western blots of Phospho-Akt (P-Akt) and GAPDH (red) and total Akt (green) from homogenates of PC12 cells cultured in the presence of BzATP for 16 h, or 16 h after Hsp90^NY intracellular delivery. Untreated PC12 cells (Ctr) or cell incubated with Chariot alone (Ch) were used as controls. MW: molecular weight marker. Bottom graph, quantitation of the infrared fluorescence signal ratio of P-Akt versus total Akt. The columns represent the mean ± SD (n = 3, individual values show as dots). *p < 0.05 vs control by Kruskal-Wallis test followed by Dunn's multiple comparison test. B-D) PC12 cells were transduced with an adenovector expressing constitutively active AKT and GFP (caAkt) or GFP alone (vector), shRNA to PTEN (shRNA PTEN) or a scramble shRNA (shRNA Ctr), or incubated with the PTEN inhibitor VOOH-PIC (2 μM) before peroxynitrite treatment (ONOO⁻) (B), release of nitrated Hsp90 (Hsp90^NY) (C), or incubation with BzATP (1 μM) (D), and cell viability assessed 24 h post-treatment. The columns represent the mean ± SD (n = 3–6 with 3–8 replicates). *p < 0.001 vs ROA or control, ** vs ONOO⁻, Hsp90^NY, or BzATP 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.)