Fig. 4. FOXO4-DRI counteracts Doxorubicin-induced senescence and chemotoxicity in vivo.
A) SA-β-GAL assay to detect senescence in IMR90 7d after 2× treatment (1d in between) with 0.1 M Doxorubicin. B) Immunofluorescence for p16ink4a and FOXO4 in control or Doxo-senescent IMR90. See also Fig. S4B. C) Quantification of the % of cells positive for p16ink4a, IL1α, IL6 and FOXO4 foci after Doxorubicin-induced senescence. D) Viability assay comparing the effect of FOXO4-DRI on control and Doxo-senescent cells in vitro. SI determined as in Fig. 3A. E) Viability assay comparing ABT-737 vs. FOXO4-DRI on Doxo-senescent cells. F) Same as in E, but both added 3× at 1/3 the final concentration. See also Fig. S4C. G) Viability assay comparing effects of incubation of FOXO4-DRI for various time points prior, during or after Doxorubicin exposure (blue line) vs. cells already induced to senesce by Doxorubicin (green boxes). M=Mock. H) Representative bioluminescence image and quantification of p16-driven senescence (RLUC) in p16∷3MR mice (See Fig. S4A+D), treated as indicated with Doxorubicin, followed by FOXO4-DRI or Mock. I) Timeline of experiments in J-N. Doxorubicin: 2× i.p. at 10mg/kg. FOXO4-DRI: 3× i.v. at 5mg/kg, every other day (day 1, 3 and 5). J) Quantification of the % change in body weight of Doxorubicin-exposed mice treated with FOXO4-DRI or PBS, respectively. K) Quantification of the number of liver cells with ≥ 10 FOXO4 foci after Doxorubicin-exposure and treatment with PBS or FOXO4-DRI. L) Visualization and quantification of the % of liver cells from the mice in (K) expressing IL-6. M) Quantification of the Doxorubicin-induced increase in plasma AST levels as marker for liver damage. N) Quantification of the effects of PBS or FOXO4-DRI on Doxorubicin-induced plasma levels of AST and Urea as markers for liver and kidney damage, respectively. See also Fig. S4E+F.