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

Figure 6. Genetic and pharmacological inhibition of AKT prevents short telomere-induced senescence.

(A) Heterozygous mutation of zTOR counteracts telomere-shortening-induced Akt activation, leading to inhibition of p15/16 expression. Western blot analysis of AKT-P and (B) RT-qPCR analysis of p15/16 mRNA levels in 13-month-old gut of WT, tert-/-, ztor+/-and tert-/- ztor+/- fish (N = 3 fish). (C-F) Second generation (G2) tert-/- mutant larvae with extremely short telomeres show phenotypes associated with premature aging, as described in Figures 1, 2 and 3. (C) Representative images of SA-β-GAL staining of WT and second generation (G2) tert-/- mutant four dpf larvae. (D) RT-qPCR analysis of p15/16 mRNA levels (N = 6), E) Western blot analysis of AKT-P, SOD2, p15/16 (N = 4) and (F) ROS levels measurements determined by DCFDA assay (N = 3). G) Survival curve of G2 tert-/-upon NAC (40 µM from day 6 to 10) treatment (WT N = 31; WT+NAC N = 27; G2 tert-/- N = 61; G2 tert-/- +NAC N = 36 fish; ** p-value<0.01; ** p-value<0.01 using Log-Rank test). (H-J) Pharmacological inhibition of AKT rescues telomere-shortening induced p15/16 expression. (H) Experimental scheme of pharmacological inhibition of AKT in G2 tert-/-. (I) Western blot analysis of AKT-P and p15/16 and (J) RT-qPCR analysis of p15/16 mRNA levels of G2 tert-/- and WT treated with AKT inhibitor. All RT-qPCR graphs are representing mean ± SEM mRNA fold increase after normalisation to rpl13a gene expression levels (* p-value<0.05; ** p-value<0.01, using t-test).

Figure 6—source data 1. Real-time qPCR data of p15/16, as plotted in Figure 6B.
Figure 6—source data 2. Real-time qPCR data of p15/16, as plotted in Figure 6D and J.
Figure 6—source data 3. ROS levels measurements, as plotted in Figure 6F.
Figure 6—source data 4. Survival analysis upon NAC treatment, as plotted in Figure 6G.

Figure 6.

Figure 6—figure supplement 1. ztor haploinsufficiency is not sufficient to suppress tissue defects in tert-/- zebrafish.

Figure 6—figure supplement 1.

The absence of one copy of the ztor gene is not sufficient to rescue the morphological defects observed in the tert-/- at 13 months of age. (A and B) Representative haematoxylin and eosin-stained sections of gut (scale bar = 40 µm) (A) and testis (scale bar = 25 µm) (B) from 13-month-old WT, tert-/-, ztor+/- and tert-/- ztor +/- siblings (N = 3 fish each).
Figure 6—figure supplement 2. Pharmacological administration of ROS scavengers increases survival of G2 tert-/-.

Figure 6—figure supplement 2.

Survival curve of G2 tert-/- upon MitoTempo (10 µM from day 3 to 5) treatment. (WT N = 26; WT+MitoTempo N = 35; G2 tert-/- N = 35: G2 tert-/- +MitoTempo N = 39 fish; * p-value<0.05 using Log-rank test).
Figure 6—figure supplement 2—source data 1. Survival analysis upon MitoTempo treatment, as plotted in Figure 6—figure supplement 2.