1. Volume 16, Issue 5, Pages 725-736 (December 2004)
DNA Damage-Induced Downregulation of Cdc25C Is Mediated by p53 via Two Independent Mechanisms 
Selvon St. Clair, Luciana Giono, Shohreh Varmeh-Ziaie, Lois Resnick-Silverman, Wen-jun Liu, Abhilash Padi, Jayasri Dastidar, Andrea DaCosta, Melissa Mattia, James J. Manfredi 
Molecular Cell 
Volume 16, Issue 5, Pages (December 2004)
DOI: /j.molcel

2. Figure 1
Downregulation of Cdc25C Protein and mRNA in Response to DNA Damage Is p53 Dependent
(A) MCF7 cells were treated with the indicated amounts of doxorubicin for 24 hr. Cell lysates were immunoblotted, and corresponding dishes were subjected to flow cytometry.
(B) The indicated HCT116 derivatives were treated with 0.5 μg/ml of doxorubicin for the times indicated. Cell lysates were immunoblotted, and corresponding dishes were subjected to flow cytometry (top) or to RT-PCR analysis (bottom).
(C) EJp53 cells were subjected to tetracycline withdrawal for the indicated times, and cell lysates were immunoblotted (top). “C” indicates extract from 293T cells after transient transfection with a Cdc25C expression plasmid. Corresponding dishes of EJp53 cells
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
The Level of cdc25C Expression Contributes to a DNA Damage-Induced Cell Cycle Checkpoint; p53 Represses via a Response Element from the Human cdc25C Promoter in a DNA Binding-Dependent Manner
(A) Four stably transfected clones of MCF7 cells were treated with 10 Gy of ionizing radiation and subjected to flow cytometry. One clone (neo) was derived from transfection with a plasmid conferring G418 sulfate resistance alone, whereas three clones (cl 3, cl 5, and cl 13) were isolated after transfection with an expression plasmid for human Cdc25C. Values are the average of three independent experiments. Bars indicate standard deviation. Immunoblotting for Cdc25C is also shown.
(B) Two stably transfected clones of HT1080 cells were isolated after transfection with an expression plasmid for a fusion protein between GFP and human Cdc25C. Cell lysates were immunoblotted (left). The indicated cells were treated with 0.5 μg/ml of doxorubicin for 48 hr and subjected to flow cytometry. The average of three independent flow cytometry experiments is shown for the HT1080 clones. Bars indicate standard deviation (right).
(C and D) Indicated cells were transfected with luciferase reporters containing various inserts upstream of the minimal adenovirus E1b promoter. Fold activation was calculated as the increase relative to each reporter in the presence of pCMV. Values are the average of three independent experiments performed in duplicate. (C) Cells were transfected with 200 ng of pCMV, pCMV-p53wt, or pCMV-p53V143A. Corresponding 100 mm dishes were transfected with pCMV (5 μg), pCMV-p53wt (1 and 5 μg), or pCMV-p53V143A (1 and 5 μg) and subjected to immunoblotting. (D) Cells were transfected with 100 ng of either pCMV (−p53) or pCMV-p53wt (+p53). The insert −186/−125(mut) is 61 bp derived from the cdc25C promoter that had the fourth position of each pentamer in the p53 site changed to a T residue. An EMSA was performed using an oligonucleotide corresponding to the 5′ site from the p21 promoter as radiolabeled probe. 5 ng of purified p53 was incubated alone, in the presence of 4 μl of mAb 421, a 10-, 30-, or 50-fold excess of unlabeled p21 5′ site, or a 100-, 200-, or 500-fold excess of each of the indicated unlabeled competitors.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
A 2 bp Substitution Outside the p53 Site Does Not Affect p53 Binding but Inhibits p53-Dependent Repression and Binding to Sp1
(A) Saos-2 cells were transfected with reporters containing various inserts in the presence of 0, 50, 200, or 600 ng of pCMV-p53wt, with total DNA brought to 600 ng with pCMV. The indicated values are the average of four independent experiments performed in duplicate.
(B) EMSA was performed using an oligonucleotide containing the consensus sequence for Sp1 binding as radiolabeled probe. In the left panel, probe was incubated in the absence of extract or in the presence of HeLa nuclear extract alone, in the presence of anti-Sp1 antibody, or in the presence of a 100-fold excess of the indicated unlabeled competitors. Probe was also incubated with purified Sp1 alone or in the presence of anti-Sp1 antibody. In the middle panel, probe was incubated with purified Sp1 alone or in the presence of a 100-fold excess of the indicated unlabeled competitors. In the right panel, probe was incubated in the absence or in the presence of HeLa nuclear extract alone, in the presence of anti-Sp1 antibody, or in the presence of a 25-fold excess of the indicated unlabeled competitors. The position of the Sp1-DNA complex is shown by the arrow, while that of the supershifted Sp1-DNA-antibody complex is indicated by the arrowhead. Two additional protein-DNA complexes are observed and are indicated by asterisks.
(C) Drosophila SL2 cells were transfected with the indicated reporters. Cells were transfected in the presence of 0, 10, 50, or 100 ng
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
A CDE/CHR Element in the cdc25C Promoter also Mediates p53-Dependent Repression; Repression via Either the p53 Binding Site or the CDE/CHR Occurs at Physiological Levels of p53 in a p21-Independent Manner
(A–D) Cells were transfected with luciferase reporters containing the indicated promoters. The cdc25C minimal promoter is represented by the insert −20/+56, whereas the −20/+56 sequence with an “X” is a mutated version of the minimal promoter. Values are the average of three independent experiments. Bars indicate standard deviation. Cells were transfected with increasing amounts (0, 50, or 75 ng) of pCMV-p53wt (A and B) or pCMV-p21 (C), with total DNA brought to 75 ng with pCMV, or were incubated in the presence or absence of tetracycline for 24 hr (D).
(E) Immunoblotting was performed on lysates from H1299 cells, transfected with either 5 μg of pCMV or pCMV-p53wt; from HCT116 cells treated with 0.5 μg/ml doxorubicin; or from EJ-p53 cells in the presence or absence of tetracycline after 24 hr.
(F) EJp53 cells were transfected with either control or p21 siRNA oligonucleotides, incubated in the presence or absence of tetracycline for 24 hr, and subjected to immunoblotting or RT-PCR analysis.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
The CCAAT Elements in the cdc25C Promoter Are Not Targets for p53-Dependent Repression at Physiological Levels of p53; p53 Associates with the cdc25C Promoter in Cells
(A and B) Cells were transfected with luciferase reporters containing the indicated promoters. The CCAAT elements are represented by the black boxes, the gray box represents the p53-response element, the white boxes represent the CDE/CHR, and the small white box represents the GC elements. −155/−125 (2×) contains two copies of the p53 binding site. Values are the average of three independent experiments performed in duplicate. In (A), H1299 cells were transfected with increasing amounts (0, 50, or 75 ng) of pCMV-p53wt, with the total DNA brought to 100 ng with pCMV. In (B), EJp53 cells were incubated in the presence or absence of tetracycline for 24 hr.
(C) EMSA was performed using an oligonucleotide corresponding to the 5′ site from the p21 promoter as radiolabeled probe. 50 ng of purified p53 was incubated as indicated, either alone, in the presence of monoclonal antibody mAb 1801, a 10-, 20-, or 50-fold molar excess of unlabeled p21 5′ site, or a 100-, 200-, or 300-fold molar excess of each of the indicated unlabeled competitors. The arrow indicates the position of the p53-DNA complex, while that of the supershifted p53-DNA-antibody complex is indicated by the bracket.
(D) H1299 cells were transfected with either pCMV or pCMV-p53wt and subjected to chromatin immunoprecipitation analysis with the indicated PCR primers.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
A Mutant p53 Protein Retains the Ability to Interact with the p53 Binding Site in the Element from the cdc25C Promoter but Fails to Mediate Repression
(A) Saos-2 cells were transfected with reporters containing the shown inserts in the presence of 0, 10, 25, 50, or 100 ng of the indicated expression plasmids, with total DNA brought to 100 ng with pCMV (top). Corresponding 100 mm dishes were transfected with 5 μg of the indicated expression plasmid, and whole-cell lysates (10, 20, or 40 μg) were immunoblotted. H1299 cells were transfected with the reporter containing two copies of the p53 binding site in the presence of 0, 50, 75, or 100 ng of the indicated expression plasmid (bottom). Values are the average of three independent experiments performed in duplicate.
(B) H1299 cells were transfected with the indicated cdc25C minimal promoter constructs in the presence of either 100 ng of pCMV or 100 ng of the shown expression plasmid. Values are the average of three independent experiments performed in duplicate.
(C) Cell lysates from corresponding dishes were transfected with the indicated p53 expression plasmids and immunoblotted with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
p53-Dependent Repression of the cdc25C Promoter Does Not Depend on Its Ability to Upregulate p21
(A and B) Indicated cell lines were transfected with various reporters in the presence of increasing amounts of pCMV-p53wt (0, 25, 50, 75, 100 ng), with total DNA brought to 100 ng with pCMV. Values are the average of three independent experiments performed in duplicate.
(C) Cell lysates from corresponding dishes were transfected with the indicated p53 expression plasmids and immunoblotted.
(D) A schematic of the cdc25C promoter is shown with the CDE/CHR elements represented by the white boxes and the p53 element represented by the gray box, respectively. The indicated GC box overlaps the p53 binding site. p53 can directly repress the cdc25C promoter via the p53 response element in a sequence-specific manner. Alternatively, CDE/CHR elements can mediate repression of the cdc25C promoter, which occurs in both a p21-dependent and -independent manner.
Molecular Cell  , DOI: ( /j.molcel )