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. 2020 May 19;9:e54935. doi: 10.7554/eLife.54935

Figure 4. Activation of AKT in older tert-/- mutants results in FoxO1/4 translocation to the cytoplasm and reduction of mitochondria OxPhos defenses.

(A) Activation of Akt leads to the inhibitory phosphorylation of FoxO1 and FoxO4 and corresponding reduction of SOD2 expression in 9-month-old tert-/- mutants. Western blot analysis for AKT-P, total AKT, FoxO1-P, FoxO4-P and SOD2 from gut extracts of 9-month-old tert-/- mutant and WT siblings (N >= 9). Representative western blot (left panel) and corresponding normalised quantification (right panel). Data are represented as mean ± SEM. * p-value<0.05; ** p-value<0.01 using the Mann-Whitney test. (B-F) Activation of Akt in older tert-/- mutant gut enterocytes leads to the translocation of FoxO1 from the nucleus to the cytoplasm and complementary accumulation p15/16 senescence marker. Total FoxO1 and p15/16 co-immunofluorescence staining in the gut of 9-month-old tert-/- and WT siblings. (B) Representative image of 9-month-old tert-/- gut. Red arrows: low nuclear FoxO1 levels in p15/16 positive cells; White arrows: high nuclear FoxO1 levels in p15/16 negative cells; scale bar: 20 µm. Dashed lines a and b depict the regions of fluorescence intensity quantification of cells analysed in D and C, respectively. (C-D) Histograms representing fluorescence quantification of DAPI, FoxO1 and p15/16 across a p15/16 positive (dashed line b) or p15/16 negative cells (dashed line a). (E) Cell analysis: High p15/16 correlates with low FoxO1 nuclear/cytoplasmic fluorescence intensity in each gut cell of tert-/- mutants. Analysis performed per cell basis (WT N = 3; tert-/- N = 2; at least 69 cells per genotype were analysed). (F) Fish analysis: On average, 9-month-old tert-/- fish (N = 2) contain more ‘low FoxO1/high p15/16’ cells than WT siblings (N = 3). Data are represented as mean per sample. p-values were calculated using a 2-factor ANOVA test.

Figure 4—source data 1. Western Blot quantifications, as plotted in Figure 4A.
Figure 4—source data 2. Mean (nuclear/cytoplasmic) p15/16 or FoxO1 fluorescence intensity per cell, as plotted in Figure 4E.
Figure 4—source data 3. Mean (nuclear/cytoplasmic) p15/16 or FoxO1 fluorescence intensity per cell, as plotted in Figure 4F.
Figure 4—source data 4. Western Blot quantifications, as plotted in Figure 4—figure supplement 1–2.

Figure 4.

Figure 4—figure supplement 1. Activation of AKT in older tert-/- mutants testis results in SOD2 reduction.

Figure 4—figure supplement 1.

Activation of Akt leads to reduction of SOD2 expression in 9-month-old tert-/- mutants. Western blot analysis for p-Akt, total Akt, and SOD2 from testis extracts of 9-month-old tert-/- mutant and WT siblings (N >= 9). Representative western blot (left panel) and corresponding normalised quantification (right panel). Data are represented as mean ± SEM. (* p-value<0.05; ** p-value<0.01 using the Mann-Whitney test).
Figure 4—figure supplement 2. Akt pathway is not induced in young tert-/-compared to wild type.

Figure 4—figure supplement 2.

Representative immunoblot of p-Akt and SOD2 and respective quantification from gut of 3-month-old tert mutant and WT siblings (N = 3). At 3 month, no differences are observed in p-Akt and SOD2 protein levels between tert-/- and WT siblings. Data are represented as mean ± SEM (ns - not significant, p>0.05, using the Mann-Whitney test).