Figure 1 FOXO4 Is Elevated in Senescent Normal Fibroblasts and Ensures Their Viability (A) Schematic representation of the mRNA expression changes ( Figure S1 A) of the cell-intrinsic apoptosis pathway ( Tait and Green, 2010 ) between senescent and control (proliferating) IMR90 fibroblasts. Inset: immunofluorescence for PUMA, BIM, and BCL2. (B) Volcano plot comparing transcriptional regulators in senescent versus control IMR90. (See Figure S1 B for expression and p values). Dark blue: associated with apoptosis.

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Inset, left: RNA expression of the FOXO cluster. Not detectable. Right: protein levels of the FOXO cluster. FOXO1 was ectopically expressed as positive control.

(C) QPCR for changes in FOXO1, 3, and 4 mRNA after senescence-induction by 10 Gy IR. P21 Cip1 (biphasic increase), p53, and ETS2 (biphasic decrease) are included as controls. (D) Immunoblot for changes in FOXO3 and 4 protein levels after senescence-induction by 10 Gy IR. (E) The senescence-induced FOXO4 mRNA expression is successfully countered by two shRNAs. (F) Cytochrome-C release assay (left) as measure for apoptosis in the conditions of (E), quantified in a histogram (right). (G) Induction of cleaved caspase-3 after senescence induction in (mouse-specific) FOXO4-deprived wild-type or bax/bak −/− BMK cells. (H) AqueousOne viability (left) and colony density (right; see also Figure S1 C) of control and senescent IMR90 cells transduced with the short hairpins used in (E).

Figure 2 FOXO4 Localizes to Senescence-Associated PML/DNA-SCARS, which Contain Active p53 and Can Be Disrupted by FOXO4-DRI (A) FOXO4 foci and senescence-associated heterochromatin structures in senescent IMR90 (see also Figures S2 A–SI). Bottom: intensity plot (arbitrary units) of individual pixels measured by the indicated line. (B) Quantification of cells containing ≥3 FOXO4 foci in time after senescence-inducing IR. (C) FOXO4 foci in senescent cells transduced with the shRNAs against FOXO4 described in Figure 1 E. (D) Structured illumination microscopic (SIM) image of the nucleus of a senescent IMR90 cell stained for FOXO4, 53BP1, and PML. Yellow arrow: Area processed for 3D surface-rendering (insets). (E) FOXO4 and Ser15-phosphorylated p53, assessed as in (A).

Note that for FOXO4 a different antibody (Sigma) was used. (F and G) Sequence (H indicates predicted helix) and 3D structure of FOXO4 used for the design of FOXO4-DRI. The amino acids indicated in yellow in (F) are shown as yellow spheres in the displayed structure of FOXO4 (3L2C, protein databank). Green aa in (F) are not visualized in this 3D structure but are part of the FOXO4-DRI sequence. Red aa in (G) change most upon p53-interaction ( Wang et al., 2008 ).

See also Figures S2 J–S2L. (H) 1H, 15N HSQC NMR spectrum of 15N-labeled recombinant FOXO4 86-206 incubated with increasing stoichiometric equivalents of recombinant p53 (60, 120, 240, or 300 μM, respectively). (I) Experiment as in (H), but with one times or two times stoichiometric equivalents of FOXO4-DRI (300 or 600 μM, respectively). (J) Cellular uptake of FOXO4-DRI in senescent IMR90 visualized by an antibody against the HIV-TAT sequence. (K) Quantification of the number of FOXO4/PML/53BP1-DNA-SCARS in control and senescent IMR90 incubated 3 days with 25 μM FOXO4-DRI and the pan caspase-inhibitor QVD-OPH. Steam Wallet Keygen No Survey No Password more. Number of small 53BP1 foci shown as control.