Skip to main content
. 2008 Oct 3;9(12):1222–1229. doi: 10.1038/embor.2008.186

Figure 5.

Figure 5

Control of Strap stabilization. (A) U2OS cells were transfected with the indicated Strap expression vector (200 ng). Cells were treated with either vehicle or etoposide (10 μM) for 16 h before fixation and processing for immunofluorescence. Strap was detected using anti-HA and DAPI staining was used to visualize nuclei; n=5. (B) U2OS cells were transfected with the indicated Strap expression vector (WT and S221A; 500 ng) and treated with etoposide (+; 10 μM) for 16 h before collection. Cell extracts were loaded on the basis of β-galactosidase activity and Strap was detected using anti-HA. The fold increase in wild-type Strap levels on DNA damage was about twofold; n=4. (C) (a) U2OS cells were transfected with pBB14 (Us9-GFP; 500 ng) as an internal transfection marker and the appropriate expression vector (500 ng). Cells were treated with or without etoposide (10 μM for 16 h) as indicated and then collected for flow cytometry analysis as described. The graph represents cell-cycle profiles of transfected cells only. (b) Immunoblot from the cells used in the FACS profiles shown in (a). Cells were loaded on the basis of β-galactosidase activity and Strap was detected using anti-HA HA11. (D) (a) U2OS cells were transfected with Strap small interfering RNA (siRNA) or control non-targeting (NT) siRNA (25 nM) for 48 h and then treated with etoposide (+; 10 μM) for 16 h. Cells were then collected for flow cytometry analysis as described. The graph represents the percentage change in G2 of Strap siRNA relative to the control NT siRNA-treated cells. (b) Immunoblot from the cells used in the FACS profiles shown in (a). Extracts were loaded on total protein content. Strap was identified by immunoblotting and β-actin was used as a loading control. (E) U2OS cells were transfected with the indicated Strap expression vectors (WT and S221A) and vector alone (500 ng), and treated with etoposide (+; 10 μM) for 16 h before collection. Extracts were loaded on total protein content. PARP was identified by immunoblotting and β-actin was used as a loading control. The asterisk indicates the cleaved PARP polypeptide; n=3. (F) Model for the dual regulation of Strap by ATM and Chk2: phosphorylation of ATM results in increased nuclear presence of Strap, whereas subsequent phosphorylation by Chk2 augments Strap stabilization. ATM, ataxia telangiectasia mutated; Chk2, Checkpoint kinase 2; DAPI, 4,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; HA, haemagglutinin; PARP, poly(ADP-ribose) polymerase; Strap, stress responsive activator of p300; WT, wild type.