Xenbase Image ID: 172001

Figure 5. Reactivation of mTOR signalling, mediated by HIF-1 function, is required when nutrient-deprived retinas increase proliferation in hypoxia. (A–D) Immunostaining for the mTOR target phospho-S6 (p-S6) in retinas from normal-fed (A–C) or nutrient-deprived (C–E) animals kept in normal oxygen concentration (A,D), under hypoxia (B,E) or under hypoxia and treated with Echinomycin (C,F). Incubation of nutrient-deprived retinas in hypoxia restores p-S6 staining in the CMZ. Echinomycin treatment interferes with the increase of p-S6 staining in the CMZ of nutrient-deprived hypoxic retinas. Scale bars = 50 µm; n = 3. In each condition, a minimum of 8 retinas was examined. (G) Western blot of retinas dissected from normal-fed or nutrient-deprived normoxic or hypoxic animals, probed for phospho-S6 and α-tubulin. At least 22 retinas were taken for each condition, and all conditions in each experiment have the same number of retinas (n = 10). (H) Quantification of the Western blot shown in (G). Phospho-S6 levels, normalized to tubulin, are significantly reduced upon nutrient deprivation, unaffected by hypoxia alone, and increase to 40% of control levels upon nutrient deprivation in hypoxia. In Western blot quantification, error bars represent standard deviations between 10 independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, *** p-values < 0.001; n = 10. (I) Quantification of EdU incorporation in retinas from normal-fed or nutrient-deprived animals kept in normoxia, hypoxia or incubated with Rapamycin under hypoxia. Nutrient-deprived retinas no longer resume their proliferation in hypoxia when mTOR signalling is blocked by Rapamycin. Error bars represent standard deviations between two independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, *** p-values < 0.001; n = 2. For each condition, between 7 and 15 retinas were quantified. Image published in: Khaliullina H et al. (2016) © 2016 by the authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Experiment + Assay Source Phenotypes and Disease Xl Wt + nutrient deprivation + hypoxia + Echinomycin + NF 41 + 24h Anatomical Phenotype abnormal protein phosphorylation Xla Wt + Echinomycin + NF38 Anatomical Phenotype abnormal protein phosphorylation Xla Wt + nutrient deprivation + NF38 Anatomical Phenotype abnormal protein phosphorylation Larger Image Printer Friendly View

Return to previous page