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. Author manuscript; available in PMC: 2018 Mar 23.
Published in final edited form as: Cell. 2017 Mar 23;169(1):132–147.e16. doi: 10.1016/j.cell.2017.02.031

Fig. 2. FOXO4 localizes to senescence-associated PML/DNA-SCARS, which contain active p53 and can be disrupted by FOXO4-DRI.

Fig. 2

A) FOXO4 foci and Senescence-Associated Heterochromatin Structures in senescent IMR90 (See also Fig. S2A–I). 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 1E). 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 2A. Note that for FOXO4 a different antibody (Sigma) was used. F+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 Fig. S2J–L. H) 1H,15N HSQC NMR spectrum of 15N-labelled recombinant FOXO486–206 incubated with increasing stoichiometric equivalents of recombinant p53 (60, 120, 240 or 300 μM, respectively). I) Experiment as in H), but with 1× or 2× 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 3d with 25 M FOXO4-DRI and the pan Caspase-inhibitor QVD-OPH. # of small 53BP1 foci shown as control. Only infrequently FOXO4 foci were visible in control cells. L) Schematic representation of the p21CIp1 (CDKN1a) promoter in which the canonical FOXO target sequence is flanked by two p53 binding sites. M+N) Quantification of nuclear p21Cip1 intensity of senescent IMR90 treated as in K). N) Left: Immunoblot of senescent IMR90 cells incubated for the indicated time points with FOXO4-DRI and processed for Ser15-phosphorylated and total p53. Middle: Nuclear exclusion of pSer15-p53 in cell treated as in K+M). Right: Quantification of pSer15-p53 foci per nucleus of senescent IMR90.