Effects of Humanin addition on the effects caused by EndoG deficiency in cell proliferation, ROS production and signal transduction. A) Equal number of Endog+/+ and Endog−/− mouse-derived skin fibroblasts were seeded. At time zero, cell number in replicate plaques was counted. DMSO (Control) or 100 nM Humanin (HN) diluted in DMSO were added in duplicate plates/treatment and cells were counted after 48 h. Data are expressed as the number of cell cycles completed in 48 h. All values from 5 independent experiments in duplicates are plotted. B) MitoSOX™ fluorescence was quantified by flow cytometry in preparations of the same experimental groups as in (A). Eight experiments for controls and four experiments for HN treatment in duplicates are plotted. C) Western Blot of pAkt, total Akt and EndoG was performed with cultures of primary skin fibroblasts of 6 Endog+/+ and 6 Endog−/− adult mice (NB: Naphthol blue staining of the membrane). After densitometric quantification of pAkt and Akt Western Blot bands, the ratio was calculated for each sample and plotted in the graph at right. Data in all the graphs represent individual values, mean ± SD (Endog+/+, blue dots; Endog−/−, red dots). Two-way ANOVA was used to analyze the influence of Endog expression and Humanin treatment and the interaction between them in cell proliferation, ROS production and Akt phosphorylation. Ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)