A, A549 cells were treated with 1 μg/mL doxorubicin (DOX) for the indicated hours. Lysates were analyzed by immunoblotting with indicated antiboies (Left). A549 cells were treated with 1 μg/mL doxorubicin (DOX) for the indicated hours and treated with or without MG132 for 3 hr. Lysates were immunoblotted with the indicated antibodies (Right).
B, Indicated cells were treated with 1 μg/mL doxorubicin (DOX) for the indicated hours. Cell lysates were immunoblotted with the indicated antibodies. The protein levels of COP1 in response to DNA damage at the different time points were determined (Right).
C, HCT116 cells were knocked down with two specific 14-3-3σ shRNA (1 & 2) or control Luciferase shRNA (C). Lysates were immunoblotted with anti-COP1, and anti-14-3-3σ (Left). 293T cells were transfected with the indicated expression vectors. Equal amounts of cell lysates were immunoblotted with the indicated antibodies (Right).
D, 293T cells were transfected with the indicated plasmids. Cells were treated with or without proteasome inhibitor MG132 before lysates were collected. Lysates were immunoblotted with the indicated antibodies (Left). Indicated cells were treated with cycloheximide (CHX) for the indicated hours. Cell lysates were immunoblotted with indicated antibodies. The protein levels of COP1 over time are shown (Right).

A, 293T cells were cotransfected with the indicated plasmids and increasing amounts of Flag-14-3-3σ. The cells were treated with MG132 for 6 hr before harvesting, and the ubiquitinated COP1 was immunoprecipitated with anti-Myc and immunobltted with anti-HA. Equal amounts of whole cell lysate (WCL) were immunoblotted with the indicated antibodies.
B, Indicated cells were treated with MG132 for 6 hr before harvesting. Polyubiquitinated COP1 was detected by immunoprecipitating with anti-COP1, followed by immunoblotting with anti-ubiquitin.
C, HCT116 cells were knocked down with two specific 14-3-3σ shRNA (1 & 2) or control Luciferase shRNA (C). Lysates were immunoprecipitated with anti-COP1 and immunoblotted with anti-ubiquitin.
D, Flag-COP1 and Flag-COP1 RING mutant (C136S, C139S) proteins were made using an in vitro Transcription/Translation system (TNT) and then incubated with the indicated purified GST proteins. Polyubiquitinated COP1 was examined by immunoprecipitating with anti-Flag, followed by immunoblotting with anti-ubiquitin (middle panel). The bottom panel shows the input of GST proteins (Coomassie Blue staining).

A, The number of live HCT116/Myc-COP1 cells, HCT116/vector control cells, COP1-expressing cells infected with Ad-β-gal or Ad-14-3-3σ, was estimated by MTT assay every day for 7 days. Optical density at 570 nm (OD570) absorbance of each group is shown. This experiment was done in triplicate. Error bars represent 95% confidence intervals.
B, HCT116/Myc-COP1 cells were infected with Ad-β-gal or Ad-14-3-3σ on day 1. Infected cells were treated with or without 10 Gy IR on day 2. Live cells were detected as dye-excluding cells and counted after trypan blue staining on day 4. The bar graph shows the percentage of live cells. Error bars represent 95% confidence intervals.
C, Indicated cells were analyzed for cell cycle distribution using fluorescence-activated cell sorting (left panel). HCT116/Myc-COP1 cells were left uninfected (-) or infected with Ad-β-gal or Ad-14-3-3σ, followed by fluorescence-activated cell sorting for cell cycle analysis. The percentages of representative cells in each phase of the cell cycle are shown for each treatment group (right panel). This experiment was done in triplicate.
D, Indicated cells were analyzed for foci formation. HCT116/Myc-COP1 cells infected with Ad-β-gal or Ad-14-3-3σ were subjected to foci formation (left panel). The bar graphs show the average of the foci numbers in these cells. Error bars represent 95% confidence intervals. HCT116/Myc-COP1 and HCT116/vector cells were analyzed in a soft agar assay, as were HCT116/Myc-COP1 cells infected with Ad-β-gal or Ad-14-3-3σ (right panel). The average number of colonies per plate is represented. Error bars represent 95% confidence intervals.

A, Protein levels of COP1 and 14-3-3σ were analyzed by immunoblotting in malignant human breast cancer specimens. The integrated optical intensity of COP1 was plotted against that of 14-3-3σ. Spearman rank order correlation was used to demonstrate the inverse correlation between COP1 and 14-3-3σ protein levels (p < 0.001). Spearman correlation=−0.61.
B, Pancreatic cancer specimens were immunostained with anti-14-3-3σ and anti-COP1. Two representative cases of the 121 examined are shown. Consecutive tissue sections from tumor case I showed a low level of 14-3-3σ but a high level of COP1. In contrast, consecutive sections from tumor case II showed abundant 14-3-3σ but scanty COP1 (original magnification, 200×). Scale bar, 50 μm.

A, A549 cells were treated with 1 μg/mL doxorubicin overnight. Before harvesting, cells were treated with MG132. Equal amounts of cell lysates were immunoprecipitated with either rabbit IgG (as control) or anti-COP1, and immunoblotted with anti-14-3-3σ (upper panel). Lysates were also immunoprecipitated with the indicated antibodies and immunoblotted with anti-COP1 (bottom panel). WCL: whole cell lysate.
B, ³⁵S-labeled COP1 proteins were translated in vitro and incubated with either GST- or GST-14-3-3σ for binding assay. The bottom panel shows the input of GST proteins analyzed by Coomassie Blue staining. PD: pull down.
C, Bacterially purified Flag-COP1 domains were incubated with GST-14-3-3σ, and then was subjected to GST pull-down (PD) followed by immunoblotting using anti-Flag. Specific interaction of COP1 domains with 14-3-3σ is indicated by arrows. The asterisks indicate non-specific bands.

A, 293T cells were cotransfected with the indicated plasmids. Lysates were immunoblotted with the indicated antibodies.
B, H1299 cells were cotransfected with the indicated plasmids. Cells were treated with MG132 before harvesting. The polyubiquitinated p53 were immunoprecipitated with anti-p53 and immunoblotted with anti-HA. Lysates were also immunoblotted with the indicated antibodies.
C, p53^−/−MDM2^−/− MEF cells were cotransfected with the indicated plasmids. Cells were treated with MG132 before harvesting. Polyubiquitination of p53 was detected by immunoprecipitation with anti-p53 and immunoblotting with anti-ubiquitin. Lysates were also immunoblotted with the indicated antibodies.
D, The mRNA levels of the indicated p53 target genes, including CDKN1A, SFN, PUMA, and BAX, were detected by real-time PCR in indicated cells stably expressing the control vector, Myc-COP1, or RFP-COP1 and normalized to GAPDH mRNA levels. Cells were also infected with the indicated Ad-β-gal (control) or Ad-14-3-3σ. The expression levels of the indicated p53 target genes were measured, and the data are presented as a heat map (Left). Statistic analysis for the heap map using Student’s t-test, p<0.05. The BDS2-3X-luc reporter containing a p53-responsive element was transfected with the indicated plasmids. Relative luciferase activity was shown (Right). Error bars represent 95% confidence intervals.

A, Myc-COP1-overexpressing HCT116 stable transfectants and HCT116/vector control transfectants were injected subcutaneously into the flank regions of female nude mice. Tumor volumes were monitored for 40 days. Tumor growth curves are shown (left panel). Error bars represent 95% confidence intervals. Tumors were dissected and weighed 40 days after cell injection (middle panel). Bar graph shows tumor weights. Error bars represent 95% confidence intervals. The photograph shows size of representative tumors (right panel).
B, Myc-COP1-overexpressing HCT116 stable transfectants were infected with Ad-14-3-3σ and Ad-β-gal. Cells were injected subcutaneously into the flanks of nude mice. Tumor growth curves were plotted and tumor weights and sizes were measured in the same manner as in (A). Error bars represent 95% confidence intervals. Tumors were dissected and weighed (bar graph) 40 days after injection. Error bars represent 95% confidence intervals. The photograph shows size of representative tumors obtained.
C, Serial tumor sections from (B) experiment were immunostained with anti-cleaved caspase-3 (left panel), anti-Ki67 (middle panel), and anti-p53 (right panel). Bar graphs show the percentage of cells with positive signals from each staining. Error bars represent 95% confidence intervals. Scale bar, 50 μm.