a, HUVECs were seeded in 6-well plates and 24 h later treated with 10 uM Etoposide (Etopo) or 1 uM Doxorubicin (Doxo) for 24 h. After incubation, cells were collected and 2 ug of total protein were examined by Western blotting with anti-Ku70 (N3H10) and anti-actin antibodies. b, HUVECs were plated onto six-well plates at 1.2×10⁵ cells per well and transfected with the indicated siRNAs as described in Materials and Methods. Results from 24 h and 48 h post-transfection are shown. c, Forty-eight h after siRNA transfection, HUVECs were treated with 10 uM Etoposide for 24 h and 48 h. Apoptosis was detected by nuclear fragmentation using Hoechst-dye nuclear staining. Data shown are averages (± S.E.) (n= 300 cells per condition in triplicate) of three independent experiments. d MEFS (ku70^+/+) were seeded in 6 well dishes and treated with 1 uM Doxorubicin for the times shown in the figure. Cells were then collected and Western blot analysis was performed with anti-Mdm2, anti-p53, anti-Ku70 and anti-Actin antibodies. e, HUVECs were seeded in 6-well plates and 24 h later treated with 1 uM Doxorubicin for the times shown in the figure. After incubation at specific times, cells were collected and examined by Western blotting with anti-Ku70, anti-Hdm2, anti-p53 and anti-actin antibodies.

a, Nutlin-3 inhibits Ku70-Hdm2 interaction. 50 ng of recombinant Hdm2 were incubated with beads preabsorbed with Hdm2 antibody, for 2 hours. After incubation, 50 ng of recombinant Ku70 (Enzymax) were added in the presence of 20 uM Nutlin-3 when indicated. After additional 2 h incubation, beads were washed and samples were analyzed by Western blotting using anti-Ku70 (H308) or anti-Hdm2 (2A10) antibodies. b, Nutlin-3 inhibited Doxorubicin-induced Ku70 proteolysis. Cells prepared as in (b) were lysed and 2 ug of total protein were analyzed by Western blotting using anti-Ku70 (N3H10) and anti-actin antibodies. c, Nutlin protected HUVECs from Doxorubicin-induced apoptosis. 1×10⁵ cells were seeded in 6-well dishes. Twenty-four h later cells were treated with 5 uM Nutlin and after 30 minutes, 1 uM Doxorubicin was added to corresponding wells. Apoptosis was detected twenty-four h later by nuclear fragmentation using Hoechst nuclear staining. Data shown are averages (± S.E.) of triplicated samples (300 cells per condition in triplicate).

a, Immunohistochemical analysis. HUVECs were seeded on gelatin-coated 35 mm dishes. Cells were serum starved for 15 minutes and treated with VEGF (100 ng/ml) for 2 h. Doxorubicin was then added for 2 h and immunohistochemistry was performed as previously described. After fixation of samples in 4% PFA for 10 minutes and permeabilization with 0.2% Triton X-100, the cellular localization of Hdm2 was detected using a monoclonal antibody against Mdm2 (IF2). After extensive washing in PBS, the samples were further incubated with Texas-Red- conjugated goat anti-mouse IgG and examined under a fluorescent microscope. Arrows identify cells with Hdm2 nuclear localization. b, Quantitative analysis of subcellular localization of Hdm2 detected by immunocytochemistry. In each sample, at least 100 cells were analyzed. Data represent averages (± S.E.) of three independent experiments performed using duplicated samples

a and b, Akt did not show cytoprotection in Ku70 siRNA treated cells. HeLa cells were plated onto six-well plates at 1.2×10⁵ cells per well and transfected with the Ku70 siRNA (SiKu70) or scrambled control RNA (Scr) as described 35. Twenty-four h after transfection, cells were infected with adenovirus expressing Myc-Myr-Akt (constitutively active) andincubated additional 24 h. Then, cells were treated with 25 uM Etoposide (Etopo) (a) or 200 nM staurosporin (STS) (b) for 16 h, and apoptosis induction was determined by Hoechst-dye nuclear staining. Data represent averages (± S.E.) (300 cells per sample were counted) of triplicated experiments. c, Ku70 siRNA successfully knocked down Ku70 in HeLa cells. Ku70 siRNA (SiKu70) and Myc-Akt were expressed in HeLa cells as described in (a), and Western blotting analysis was performed with anti-Ku70, anti-Bax, anti-Bcl-2, anti-Myc and anti-actin. d, Akt did not show significant cytoprotective activity in Ku70 null MEFs. Wild type and ku70−/− MEFs (8×10⁴ cells/well) were seeded in 12-well plates. Twenty-four h later cells were infected with adenovirus expressing Myc-Myr-Akt. The following day cells were treated with 10 uM Etoposide for additional 12 h and apoptosis was analyzed as described in Fig. 1a. e, Expression levels of Ku70, Bax, and Bcl-2 of wild type and ku70−/− MEFs. Wild type and ku70−/− MEFs were analyzed by Western blotting with anti-Ku70 (N3H10), anti-Hdm2 (2A10), anti-p53, anti-Bax (N20), anti-Bcl-2, and anti-actin antibody.

a, Hdm2 overexpression decreased Ku70 levels. HUVECs were transfected with pCMV vector, pCMVHdm2-wt or pCMVHdm2-C464S (dead ligase) and cultured for 16 h. Cells were then collected and analyzed by Western blot using anti-Ku70, anti-Hdm2 and anti-Actin antibodies. b, HEK293 cells were transfected with pCMVHdm2-wt and HA-tagged Ubiquitin-wt constructs as indicated. Twenty-one hours later cells were treated with 5 uM MG132 for 3 h; cells were then collected and lysed in RIPA buffer. Immunoprecipitation with anti-Ku70 antibody (H308) was performed as indicated in Materials and Methods, and Western blot was done with anti-Ku70 (N3H10) and anti-HA antibody. Mouse IgG was used as control for the immunoprecipitation. c, Ku70 level in mdm2+/+ and mdm2−/− MEFs. Mdm2−/− (p53−/−) and mdm2+/+(p53−/−) MEFs were seeded in 10-cm dishes. Twenty four hours later, cells were harvested and lysed with RIPA buffer. Western blot analysis was then performed with anti-Ku70 (N3H10 antibody). Mdm2 antibody was used to confirm Mdm2 levels. d, In vitro ubiquitination assay of Ku70 by Hdm2. Reaction volumes were prepared as indicated in Material and Methods and incubated for 2 h at 37°C in the presence or absence of 10 uM unlabeled ubiquitin. Reactions were quenched with 50 ul of Laemmli sample buffer containing 2% (v/v) B-mercaptoethanol and boiled for 5 minutes. Western blot was performed with anti-ubiquitin antibody. e, Co-immunoprecipitation of Hdm2 and Ku70. HUVECs were collected and immunoprecipitation with anti-Hdm2 antibody was performed in Chaps buffer. Western blot analysis was done using anti-Ku70 antibody, anti-Ku80 antibody and anti-Hdm2 antibody. f, GST pull-down assay using GST-tagged Hdm2 and recombinant Ku70 was peformed using the ProFound™ pull-down GST protein:protein interaction kit (Pierce) as described by the manufacturer. Interaction was analyzed by Western blotting using anti-Ku70 antibody.

a, VEGF inhibited doxorubicin-induced apoptosis, HUVECs were serum starved for 15 min and treated with VEGF (100 ng/ml) for 2 hours. Doxorubicin was then added for additional 14 h. Apoptosis was detected by nuclear fragmentation using Hoechst-dye nuclear staining, and b, by caspase activation. Data represent averages (± S.E.) (from 3 independent experiments). c. VEGF inhibited Ku70-proteolysis induced by Doxorubicin. HUVECs were seeded in 6-cm dishes and 24 h later treated with VEGF (100 ng/ml) in serum free media for 2 h. Doxorubicin (1 uM) was then added and cells were collected after additional 16 h incubation. Western blot analysis with anti-Ku70, anti-Hdm2, anti-phospho-Hdm2 (Ser 166, Akt-dependent phosphorylation site), anti-phospho-Akt (Ser 473), anti-total Akt and anti-Actin antibodies was performed 30. This is representative of three independent experiments.

a, Akt inhibited etoposide-induced apoptosis in HeLa cells. HeLa cells were transfected with control plasmid pcDNA3 (control) or plasmid expressing HA-Myr-Akt (myr-Akt). Twenty-four h later transfection, cells were treated with 25 uM Etoposide (Etopo) for additional 16 h and apoptosis was detected by nuclear fragmentation using Hoechst nuclear staining. Data represent averages (± S.E.) of 3 independent experiments (300 cells per sample were counted). b, Akt inhibited etoposide-induced apoptosis in HUVECs. 2×10⁵ HUVECs per well were seeded in 6-well plates. Next day cells were infected using adenovirus expressing Akt constructs (Myc-Myr-Akt (constitutively active), Myc-K179M-Akt (dominant negative) or empty vector (control virus)). Twenty-four h later cells were extensively washed with Hank’s Buffer Saline Solution (HBSS) and then treated with 10 uM Etoposide (Etopo) for 24 h. Infection efficiency was almost 100% as shown in Supplemental Figure 2a. Data represent averages (± S.E.) of 3 independent experiments (300 cells per sample were counted) c, Akt inhibited etoposide-induced Ku70 level decrease in HUVECs. Cells were treated as described in (a). After the treatment, cells were collected and subcellular fractionation was performed as described in Materials and Methods. Each fraction subjected to Western blot and expression level of Ku70, Actin, and HA-Akt was determined. d, Akt inhibited etoposide-induced Ku70 level decrease in HeLa cells. Cells were treated as described in (b). Cell lysates were subjected to Western blot analysis and expression levels of Myc-Akt, phospho-GSK3, Ku70 and actin were determined.

a, Right panels: Akt maintained Ku70-Bax interaction in etoposide-treated HUVECs. HUVECs were were infected with control adenovirus or adenovirus expressing Myc-Myr-Akt. The next day, cells were treated with 20 uM Etoposide for 8 h. Cells were then lysed and Ku70 (top panel) was co-immunoprecipiatated by anti-Bax polyclonal antibody (detecting both conformationally inactive and active Bax). Activated Bax (second panel) was immunoprecipitated by 6A7 monoclonal antibody (detecting conformationally active Bax). Western blot analysis with anti-Ku70 monoclonal (top and third panels) or anti-Bax polyclonal antibody (N20) (second and fourth pannels) was performed. The levels of Ku70 and Bax in cells in each condition are shown in the third and fourth panels. Left panels: Ku70−/− MEFs were treated as described for Right Panel experiments, and active Bax was immunoprecipitated by 6A7 monoclonal Ab. Immunoprecipitated Bax was detected by Western blot using Bax polyclonal Ab. Ku70 and Bax levels in each sample are shown in the second and third panels, respectively. b, Hypothesized model for Ku70 regulation by Hdm2 and Akt. In the presence of growth/survival factor signals (Left scheme), Ku70 levels are high enough to suppress Bax activation. Akt promotes Hdm2 translocation to the nucleus and induces the targeting of p53 for degradation. In the presence of apoptotic signals without active Akt signals (Right scheme), Hdm2 remains in the cytosol and it targets Ku70 for degradation. This condition allows the release of Bax from inhibition, and Bax is activated by activators such as BH3 only proteins that are activated by p53.