Figure 1. EGC-derived 15d-PGJ2 (exerts neuroprotection against oxidative stress-induced cell death.
A, primary cultures of ENS were treated or not treated with H2O2 (200 μm; 24 h; filled and open bars, respectively) with or without (control) previous treatments with 15d-PGJ2 (1, 2 or 3 μm; 72 h). NSE released in the medium was measured by immunoradiometric assay. Values are the mean ± SEM of from 6 to 11 independent experiments (#P < 0.05 as compared to cultures without H2O2; *P < 0.05 as compared to primary cultures treated with H2O2 but without 15d-PGJ2 pretreatment; two-way ANOVA followed by Bonferroni's post hoc test). B, primary cultures of ENS were treated or not treated with H2O2 (200 μm, 24 h), with or without previous treatment with 15d-PGJ2 (3 μm; 72 h). Immunostaining was performed with anti-β-tubulin III antibody. Images are representative of 4 independent experiments. Scale bar: 100 μm. C, SH-SY5Y cells were treated or not treated with H2O2 (200 μm; 24 h; filled and open bars, respectively), with or without (control) previous treatments with different concentrations of 15d-PGJ2 (0.1, 1 or 5 μm; 24 h). Neuronal cell death was analysed by measuring the cell permeability to 7-amino-actinomycin D (% 7-AAD+ cells) by flow cytometry. Values are the mean ± SEM from four independent experiments (#P < 0.05 as compared to conditions without H2O2; *P < 0.05 as compared to conditions treated with H2O2 but without 15d-PGJ2 pretreatment; two-way ANOVA followed by Bonferroni's post hoc test). D, EGCs were transfected with shRNA PTGDS (EGC shPTGDS) or with the shRNA PTGDS inefficient construction (EGC shMOCK). Inset: different non-transformed EGCs (NT; ROG) express L-PGDS. Immunoblot analysis using antibodies against change to L-PGDS or βactin were carried out from EGC extracts. Quantitative analysis was performed by measuring band densities with ImageJ. Values are the mean ± SEM from four independent experiments (*P < 0.05 as compared to EGCs or EGC shMOCK; one-way ANOVA followed by Tukey's post hoc test). E, SH-SY5Y cells were treated or not treated with H2O2 (200 μm; 24 h; filled and open bars, respectively) after 96 h co-culture with EGCs, EGC shPTGDS or EGC shMOCK. Neuronal cell death was analysed by measuring the cell permeability to 7-amino-actinomycin D (% 7-AAD+ cells) by flow cytometry. Values are the mean ± SEM from five independent experiments (#P < 0.05 as compared to control without H2O2, *P < 0.05 as compared to H2O2-treated SH-SY5Y cells alone; †P < 0.05 as compared to SH-SY5Y cells co-cultured with EGCs or EGC shMOCK; one-way ANOVA followed by Tukey's post hoc test).