Journal: Journal of Biological Chemistry

Article Title: Differential Roles for Checkpoint Kinases in DNA Damage-dependent Degradation of the Cdc25A Protein Phosphatase

doi: 10.1074/jbc.m802474200

Figure Lengend Snippet: FIGURE 1. Phosphorylation of Cdc25A by Chk1 and Chk2 in vitro. A, GST- Chk1 and GST-Chk2 proteins purified from insect cells. Proteins were purified as described under “Materials and Methods.” Two g of each protein was subjected to SDS-PAGE and the gel-stained with Coomassie Brilliant Blue. B, GST-Cdc25C200–256 (5 g) was incubated with 0.1 g of GST-Chk1 (lane 2), 1 g of GST-Chk2, or with the appropriate volume of control buffer (lane 1) in the presence of [32P]ATP as described under “Materials and Methods.” Reac- tion mixtures were subjected to SDS-PAGE and Coomassie staining, and the dried gel was subjected to autoradiography to detect phosphorylated GST- Cdc25C200–256. C, Cdc25A or Cdc25AS76A (1 g, purified from bacteria) was incubated with 100 ng of GST-Chk1 and 1 g of GST-Chk2, conditions that provide equivalent phosphorylation of Cdc25C200–256 in the presence of [32P]ATP (see “Materials and Methods”). Reaction mixtures were subjected to SDS-PAGE and Coomassie staining, and the dried gel was subjected to auto- radiography to detect phosphorylated Cdc25A. WT, wild type.

Article Snippet: The Cdc25A antibody employed were either from Neomarkers (AB-3) or Santa Cruz.

Techniques: Phospho-proteomics, In Vitro, Purification, SDS Page, Staining, Incubation, Control, Autoradiography, Bacteria

Journal: Journal of Biological Chemistry

Article Title: Differential Roles for Checkpoint Kinases in DNA Damage-dependent Degradation of the Cdc25A Protein Phosphatase

doi: 10.1074/jbc.m802474200

Figure Lengend Snippet: FIGURE 2. Chk1, but not Chk2 phosphorylates Ser-76 in Cdc25A. A, two- dimensional tryptic peptide mapping of Cdc25A phosphorylated by Chk1 and Chk2 in vitro. Equal counts/min (3000) of Cdc25A or Cdc25AS76A phos- phorylated by GST-Chk1 or GST-Chk2 (Fig. 1C) were excised from dried gels and subjected to tryptic peptide mapping as described under “Materials and Methods.” Peptides were visualized by autoradiography. The position of a peptide absent from Cdc25AS76A is indicated with a circle (peptide b). B, an epitope recognized by an antibody against phosphoSer-76 in Cdc25A is pro- ducedinthepresenceofChk1butnotChk2.Kinasereactionswereperformed with the indicated quantity of Chk1 or Chk2 and 1 g of Cdc25A (or Cdc25AS76A) purified from bacteria. Blots were probed with anti-phospho- Ser-76(1:500)andsubsequentlystrippedandprobedwithamonoclonalanti- body against Cdc25A as a control for loading. WT, wild type.

Article Snippet: The Cdc25A antibody employed were either from Neomarkers (AB-3) or Santa Cruz.

Techniques: In Vitro, Autoradiography, Purification, Bacteria, Control

Journal: Journal of Biological Chemistry

Article Title: Differential Roles for Checkpoint Kinases in DNA Damage-dependent Degradation of the Cdc25A Protein Phosphatase

doi: 10.1074/jbc.m802474200

Figure Lengend Snippet: FIGURE 3. Chk1, but not Chk2, supports SCF-TRCP-dependent Cdc25A ubiquitination in vitro. Cdc25A ubiquitination reactions were performed as described under “Materials and Methods” in the presence of Chk1 (100 ng) or Chk2 (1 g). Reactions mixtures were subjected to SDS-PAGE, and dried gels were visualized by autoradiography.

Article Snippet: The Cdc25A antibody employed were either from Neomarkers (AB-3) or Santa Cruz.

Techniques: Ubiquitin Proteomics, In Vitro, SDS Page, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Differential Roles for Checkpoint Kinases in DNA Damage-dependent Degradation of the Cdc25A Protein Phosphatase

doi: 10.1074/jbc.m802474200

Figure Lengend Snippet: FIGURE 4. Cells lacking Chk2 retain their ability to degrade Cdc25A in a Chk1-dependent manner. A,HCT116cellsandHCT116cellslackingtheCHK2gene(HCT116CHK2/)wereeitherleftuntreatedortreated with 10 gray (Gy) before the immediate addition of cycloheximide (CHX) to block ongoing protein translation. Cells were harvested at the indicated times after IR treatment. Cells were lysed and subjected to immunoblot- ting with indicated antibodies; Cul1 is used here as a loading control. B and C, HCT116 or HCT116 CHK2/ cells were depleted for Chk1 with two distinct small interfering RNA sequences. Forty-eight hours post-trans- fection cells were treated with 10 gray before the addition of cyclohexamide. Cell lysates were resolved on SDS-PAGE and analyzed using chemiluminescent imaging and CCD detection, which allowed for the quanti- tative analysis of relative protein levels in C. In C, turnover rates for three independent experiments are shown.

Article Snippet: The Cdc25A antibody employed were either from Neomarkers (AB-3) or Santa Cruz.

Techniques: Blocking Assay, Western Blot, Control, Small Interfering RNA, SDS Page, Imaging

Journal: The Journal of Biological Chemistry

Article Title: miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks *

doi: 10.1074/jbc.M116.772392

Figure Lengend Snippet: Identification of GSK3B as a new target of miR-21 to stimulate DNA-PKcs activity. A, illustration of conserved sequences at 3′-UTR of GSK3B between mice (m) and humans (h), which match the miR-21-5p and miR-21-3p binding regions. The underlying sequences are deleted in the mutant vectors. B, the effects of the potential miR-21 binding sites at the 3′-UTR of GSK3B on luciferase activity were measured as described under “Experimental Procedures.” 293FT cells were transfected with a firefly luciferase reporter plasmid containing a partial 3′-UTR of GSK3B with the putative miR-21 binding site (WT) or deleted the key binding site (DM). Luciferase activity was assayed 24 h after transfection with miR-21 mimic (miR-21) or control (Ct) RNA and was normalized to the activity of the firefly luciferase expressed from the same pGL3-control vector, **, p < 0.01. C, wild type or miR-21 knock-in MEF cells were transfected with the GSK3B vector or CDC25A vector, or co-transfected with GSK3B/CDC25A vectors for 48 h. The cells were either exposed to IR (4 Gy for DNA-PKcs detection) or given no IR exposure, and then the cells were collected at 1 h after IR for the standard Western blotting assay. Similar results were obtained from two independent experiments. p-DNA-PKcs, phospho-DNA-PKcs. D, human 293FT cells were transfected with miR-21, GSK3B vector, or CDC25A vector or co-transfected with GSK3B/CDC25A vectors for 48 h. The cells were either exposed to IR (4 Gy for DNA-PKcs detection) or given no IR exposure, and then the cells were collected at 1 h after IR for the standard Western blotting assay. Similar results were obtained from two independent experiments. E, at 48 h after transfection as described in C, the cells were exposed to IR (4 Gy) and collected for sensitivity measurements using a clonogenic assay as described in the legend for Fig. 1. Data shown are the mean ± S.D. from triple sets of two independent experiments; ND, no significant difference. F, at 48 h after transfection as described in D, the cells were exposed to IR (4 Gy) and collected for sensitivity measurement using a clonogenic assay as described in the legend for Fig. 2. Data shown are the mean ± S.D. from triple sets of two independent experiments; ND, no significant difference.

Article Snippet: The plasmid containing mouse CDC25A was purchased from OriGene Inc., and the human CDC25A was obtained from Dr. Jiri Bartek's lab ( 43 ).

Techniques: Activity Assay, Binding Assay, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Knock-In, Western Blot, Clonogenic Assay

Journal: The Journal of Biological Chemistry

Article Title: miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks *

doi: 10.1074/jbc.M116.772392

Figure Lengend Snippet: miR-21-mediated radioresistance occurs through targeting both GSK3B and CDC25A. A, upper panel, whole cell lysates were prepared from wild type, GSK3B−/−, and cyclin D1−/− MEF cells that were treated with control RNA (CtRNA, lanes 1–3), cyclin D1 siRNA (lanes 4–6), or CDC25A siRNA (lanes 7–9). The protein levels were measured using a standard Western blotting assay. Actin was used as an internal loading control. Lower panel, cell survival fraction from 4 Gy irradiated cells. The data were mean ± S.D. from three independent experiments, **, p < 0.01. B, NHEJ or HRR efficiency was examined in MEF cells that were transfected with the reagents as described above for 24 h and then transfected with either the HRR or NHEJ reporter for an additional 24 h. The NHEJ or HRR efficiency assays were as described in the legend for Fig. 3. The data presented are the mean ± S.D. from three independent experiments. C, the protein levels were examined in human 293FT cells at 48 h after the cells were transiently transfected with GSK3B siRNA, CCND1 (cyclin D1) siRNA, or CDC25A siRNA. Actin was used as an internal loading control. D, after 293FT cells were transfected with the reagent for 24 h, the cells were transfected with the NHEJ or HRR reporter for an additional 24 h, and then the cells were collected to detect NHEJ or HRR efficiency. Data shown are the mean ± S.D. from triple seta of two independent experiments; ND, no significant difference.

Article Snippet: The plasmid containing mouse CDC25A was purchased from OriGene Inc., and the human CDC25A was obtained from Dr. Jiri Bartek's lab ( 43 ).

Techniques: Western Blot, Irradiation, Transfection

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 1. Coordinated expression of FOXM1 and CDC25A during the cell cycle progression. (A) Representative flow cytometry analysis shows cell cycle progression in U2OS cells. U2OS were synchronized with nocodazole for 16 h and then stimulated to re-enter the cell cycle by addition of medium containing 10% fetal bovine serum. Asynchronous cells (Asynch) were included as controls. Cell cycle was analyzed by flow cytometry at release (16 h Noc) and at 1, 3, 6, 12, and 18 h after release. (B) Mean percentage (6 SD) of cells in each phase of the cell cycle following 16 h nocodazole treatment and after 1, 3, 6, 12, 18 h release (N = 3). Asynchronous (Asynch) cells were included as controls. The analysis indicates that the cells at early, middle and late G1 phase are at 1, 3 and 6 h respectively, S phase is maximal at 12 h while G2/M phase occurs at 16 h Noc and 18 h after release. (C) Cells at the continuous cell cycle phases were collected and processed for western blotting analysis with antibodies against FOXM1, CDC25A, CDK1 and CDK2. FOXM1 expression increased as cells progressed from G1 through S and into G2/M, and degraded when cells exited to G2/ M (1 h). CDC25A and CDK1 exhibited a similar expression profile. CDK2 expression levels were relatively constant throughout the cell cycle. b-Actin expression was used as a loading control. doi:10.1371/journal.pone.0051277.g001

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Expressing, Flow Cytometry, Western Blot, Control

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 2. The FOXM1 transcription factor regulates CDC25A gene expression. (A) FOXM1 increased the transactivation of CDC25A promoter. U2OS cells were co-transfected with pGL3-CDC25A promoter reporter along with increasing doses of pACT-FOXM1 for 48 h. pACT vector was used as the empty vector, and pRL-SV40 expressing Renilla luciferase was used as an internal control. Data represent the mean 6 SD (N = 3). *, p,0.05; ***, p,0.001. (B) siRNA-mediated silencing of FOXM1 decreased CDC25A expression. U2OS cells were transiently transfected with siRNA pools targeting FOXM1 or non-target control for 48 h. A U2OS non-transfected control was also used. The protein expression of FOXM1 and CDC25A were detected by Western blot. b-Actin expression was used as a loading control. The western blot was repeated three times and the protein expression was analyzed by the Adobe Photoshop CS4 software (Adobe Systems, San Jose, CA). The difference of band density between the FOXM1-siRNA and control siRNA was analyzed by the paired t test. ***, p,0.001. doi:10.1371/journal.pone.0051277.g002

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Gene Expression, Transfection, Plasmid Preparation, Expressing, Luciferase, Control, Western Blot, Software

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 3. FOXM1 regulates the gene transcription of CDC25A by direct DNA binding as well as the E2F pathway. (A) Schematic diagram of CDC25A promoter showing the 39 distal end containing two E2F binding sites, the 59 proximal end including three putative FOXM1 binding sites and excluding E2F binding sites, and a 224 bp fragment containing the three putative FOXM1 binding sites. (B) FOXM1 increased the transactivation of full-length and truncated CDC25A promoter constructs, indicating both direct and indirect FOXM1 induction. U2OS cells were co- transfected with pACT-FOXM1 or pACT empty plasmid and the reporter plasmids containing full length and truncated CDC25A promoters. pRL-SV40, a Renilla luciferase report vector, was co-transfected as the control. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD (N = 5). ***, p,0.001. (C) FOXM1 recruits p300, but not PCAF or CBP, to activate CDC25A promoter. U2OS cells were co-transfected with pACT-FOXM1 (or pACT control) and pCMV-p300, or pCMV-pCAF, or pCMV-CBP (or empty vector control), along with pGL3-CDC25A promoter for 48 h. pRL-SV40 was co-transfected as the normalizing control. Data are normalized to Renilla activity, and represented as the mean 6 SD. The difference was analyzed by paired t test. ***p,0.001, D.0.05. (D) Representation of the wild-type and mutated E2F consensus binding sequences at CDC25A promoter. (E) Two E2F binding sites are required for FOXM1-activated CDC25A promoter transactivation. U2OS cells were co-transfected with pACT-FOXM1 or empty vector control and the reporter plasmids containing the indicated CDC25A promoters (wild-type or mutated E2F binding sites). Data are presented as the mean fold induction of FOXM1 activity over control 6 SD. *** p,0.001. doi:10.1371/journal.pone.0051277.g003

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Binding Assay, Construct, Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 4. FOXM1 directly binds to the putative FOXM1 consensus binding sequences located on the CDC25A promoter. (A) FOXM1 binding to CDC25A promoter was confirmed by ChIP-qPCR. Primer set A was designed to span three FOXM1 binding sites, and primer set B, targeting a region of the promoter that did not include the FOXM1 binding sites, was used as the control. Chromatin immunoprecipitations of U2OS cells were prepared using anti-FOXM1 antibody. Semi-quantitative PCR was performed using the primer sets A and B, and the PCR products were detected by electrophoresis (Top). Quantitative PCR was performed using the primer sets A and B, and the binding activity of FOXM1 to CDC25A promoter was evaluated by the fold enrichment method. (B–C) The three FOXM1 binding sites on the CDC25A promoter are functionally redundant (B) Schematic diagram showing the mutations of the three FOXM1 binding sites on the CDC25A promoter. (C) U2OS cells were transfected with pACT-FOXM1 or empty vector control, along with the indicated CDC25A promoter-luciferase reporter construct. Targeted mutation of single FOXM1 binding sites did not significantly affect CDC25A transactivation. Mutations of sites 1 and 2, sites 2 and 3, and sites 1, 2, and 3 significantly reduced the transactivation of the CDC25A-luciferase reporter. Data were normalized to Renilla luciferase activities and are presented as the mean wild-typefold induction over empty vector control 6 SD (N = 3). Data were subjected to one-way ANOVA (significance level a = 0.05) and Dunnett’s multi-comparison post-hoc tests (mutations vs. wild-type). * P,0.05. doi:10.1371/journal.pone.0051277.g004

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Binding Assay, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction, Electrophoresis, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Mutagenesis, Comparison

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 5. CDC25A regulated FOXM1 transcriptional activity through CDK1-mediated phosphorylation sites. (A) CDC25A activated the transcriptional activity of FOXM1 in U2OS cells. U2OS cells were co-transfected with pACT-FOXM1 or/and pACT-CDC25A, along with the pGL3- 66FOXM1-Luc plasmids containing 6 FOXM1 DNA binding sequences, and pRL-SV40, a Renilla luciferase reporter vector was used as the control. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD (N = 3). *** p,0.001. (B) siRNA-mediated inhibition of CDK1 blocked CDC25A-mediated FOXM1 transcriptional activity. U2OS cells were co-transfected with pACT-CDC25A, pACT-FOXM1 and pGL3-66FOXM1- Luc plasmids, or pACT-FOXM1 and pACT-CDC25A together with siRNAs against CDK1, CDK2, CDK4 or CDK6 or control siRNA, and pRL-SV40 was used as the normalizing control. Forty-eight hours after transfection, the cells were lysed, and firefly and Renilla luciferase activities were measured. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD (N = 3). Data were subjected to one-way ANOVA (significance level a = 0.05) and Dunnett’s multi-comparison post-hoc tests (CDK siRNA vs. CTR siRNA). *** p,0.001. (C) CDC25A activated FOXM1 transcriptional activity via the CDK phosphorylation sites T600, T611, and T620 and a LXL docking motif at L656. U2OS cells were co-transfected with pACT-CDC25A and pACT-FOXM1 (wild-type or the mutations of phosphorylation sites/LXL motif), along with pGL3-66FOXM1-Luc plasmids. pRL-SV40 was used as the normalizing control. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD (N = 3). Data were subjected to one- way ANOVA (significance level a = 0.05) and Dunnett’s multi-comparison post-hoc tests (mutations vs. wild-type). *** p,0.001. doi:10.1371/journal.pone.0051277.g005

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Activity Assay, Phospho-proteomics, Transfection, Binding Assay, Luciferase, Plasmid Preparation, Control, Inhibition, Comparison

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 6. CDC25A phosphatase activates FOXM1 transcriptional activity by direct protein-protein interaction. (A) CDC25A and FOXM1 proteins physically interact. The protein-protein interaction of FOXM1 and CDC25A was confirmed by co-immunoprecipitation. HEK293T cells were co-transfected with p36FLAG-CMV-14-FOXM1 (FOXM1-36FLAG) or the empty vector control p36FLAG-CMV-14, along with pCMV3Tag9-CDC25A (CDC25A-36MYC) or the empty vector control (pCMV3Tag9). Lysates were immunoprecipitated with anti-FLAG agarose resin, separated by PAGE, and electroblotted to PVDF. Western blot analysis showed that CDC25A-36MYC co-immunoprecipitates with FOXM1-36FLAG, supporting the hypothesis that these two expressed proteins interact. (B) The native protein interaction between FOXM1 and CDC25A was detected by co-immunoprecipitation. 66106 U2OS cells were lysed in MPER buffer containing 16 protease/phosphatase inhibitors. Whole cell lysates were pre-cleared with protein G sepharose and normal rabbit IgG overnight at 4uC with end-over-end mixing. The cell lysates were incubated with anti-CDC25A antibody overnight at 4uC and then with protein G sepharose for 1 h at 4uC. The resin was washed three times, and the eluted protein complex was separated by PAGE and analyzed using the anti-FOXM1 antibody by Western blotting. (C) Schematic diagram showing FOXM1 deletion constructs used to identify FOXM1 domains critical to the interaction with CDC25A. Sequences encoding the specific FOXM1 deletion constructs were subcloned in pBIND. (D) A mammalian two-hybrid assay reveals a critical role for the FOXM1 C-terminus in interactions with CDC25A. pBIND construct (FOXM1, deletion, or empty) was co-transfected with pACT construct (CDC25A or empty) and pG5luc reporter. Data represent the mean 6 SD, normalized to Renilla luciferase activities (N = 3). Co-expression of the C-terminal FOXM1 and CDC25A revealed a robust interaction. In a construct lacking the C-terminus, the interaction between FOXM1 and CDC25A was significantly diminished. *** p,0.001. (E) Schematic diagram showing the mutations of the CDK/ cyclin phosphorylation sites and LXL motif at the C-terminal of FOXM1 protein. pBIND construct (FOXM1 wild-type and FOXM1 with mutations of phosphorylation sites/LXL motifs) was co-transfected with pACT construct (CDC25A or empty) and pGL5-luc reporter. Data represent the mean 6 SD, normalized to Renilla luciferase activities (N = 3). Data were subjected to one-way ANOVA (significance level a = 0.05) and Dunnett’s multi-comparison post-hoc tests (mutations vs. wild-type). *** p,0.001. (F) Mutation of T600 and T611 diminishes FOXM1 protein association with CDC25A by co- immunoprecipitation. U2OS cells were transiently transfected with either FOXM1-36FLAG construct encoding wild-type FOXM1 (lane 1) or FOXM1 with the following mutations: T600A (lane 2), T611A (lane 3), T620A (lane 4) and L656A (lane 5). U2OS cell lysates were prepared 48 hours after transfection and FOXM1 expression in these cell lysates was detected by Western blot. The same amount of protein was co-immunoprecipitated with anti-CDC25A antibody, and the co-immunoprecipitated proteins were subjected to Western blot with an anti-FLAG antibody. The CDC25A expression in the co-immunoprecipitated proteins was used to test the efficiency of co-immunoprecipitation. The phosphorylation of T600 and T611 enhanced the protein interaction of FOXM1 and CDC25A. doi:10.1371/journal.pone.0051277.g006

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Western Blot, Incubation, Construct, Two Hybrid Assay, Luciferase, Expressing, Phospho-proteomics, Comparison, Mutagenesis

Journal: PloS one

Article Title: Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

doi: 10.1371/journal.pone.0051277

Figure Lengend Snippet: Figure 7. CDC25A phosphatase enzyme activity is required for the CDC25A-activated FOXM1 transcriptional activity and protein- protein interaction between CDC25A and FOXM1. (A) CDC25A phosphatase enzyme activity is required for the CDC25A-activated FOXM1 transcriptional activity. The C431 site in CDC25A protein was changed via site-directed mutagenesis to a serine to create a phosphatase-dead mutant. U2OS cells were co-transfected with pACT-FOXM1 and/or pACT-CDC25A (pACT-CDC25A C431S), along with the pGL3-66FOXM1-Luc plasmids containing 6 FOXM1 DNA binding sequences. pRL-SV40, a Renilla luciferase reporter vector, was used as the control. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD (N = 5). *** p,0.001. (B) A mammalian two-hybrid assay confirmed a critical role for CDC25A phosphatase activity in mediating the FOXM1-CDC25A protein-protein interaction. U2OS cells were co-transfected with pBIND-FOXM1 (or pBIND control) and one of following: wild-type pACT-CDC25A, or pACT-CDC25A C431S phosphatase-dead mutant, or pACT control. pG5-luc was used as a normalizing control. Data were normalized to Renilla luciferase activities and are presented as the mean 6 SD, (N = 5). Loss of phosphatase activity by CDC25A led to a significant reduction in the FOXM1-CDC25A interaction. ** p,0.01; *** p,0.001. doi:10.1371/journal.pone.0051277.g007

Article Snippet: The CDC25A plasmid was purchased from Origene and subcloned in-frame in the pACT expression vector at the BamHI and XbaI restriction sites or the pCMV3Tag9 (Agilent, La Jolla, CA) expression vector at the BamHI and XhoI restriction sites.

Techniques: Activity Assay, Mutagenesis, Transfection, Binding Assay, Luciferase, Plasmid Preparation, Control, Two Hybrid Assay

Journal: Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics

Article Title: miRNA-497 Negatively Regulates the Growth and Motility of Chondrosarcoma Cells by Targeting Cdc25A

doi: 10.3727/096504016x14519157902681

Figure Lengend Snippet: Figure 3. miR-497 targets the 3¢-UTR of Cdc25A. (A) The mRNA and protein expression of Cdc25A were detected by qRT-PCR and Western blot in JJ012 and OUMS-27 cells transfected with miR-497 mimic and antagomir. (B) Predicted miR-497 target sequences in the 3¢-UTR of Cdc25A. (C) Luciferase assay of cells transfected with Cdc25A-3¢UTR-wt (UTR-wt) and Cdc25A-3¢UTR-mut (UTR-mut) *p < 0.05 versus control.

Article Snippet: Transfection with 100 nM miR-497 mimic, antagomir, and negative control miRNA (RiboBio, Guangzhou China) or 1 μg CDc25A plasmid (NM_001789) (Origene, Rockville, MD, USA) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Control

Journal: Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics

Article Title: miRNA-497 Negatively Regulates the Growth and Motility of Chondrosarcoma Cells by Targeting Cdc25A

doi: 10.3727/096504016x14519157902681

Figure Lengend Snippet: Figure 4. miR-497 modulates cell cycle progression by targeting Cdc25A. (A) The mRNA and protein levels of Cdc25A were mea- sured by qRT-PCR and Western blot in JJ012 and OUMS-27 cells transfected with specific Cdc25A plasmid or control plasmid (Mock). The proliferation (B) and apoptosis (C) of cells were investigated in JJ012 and OUMS-27 cells after transfection by miR-497 mimic and Cdc25A plasmid. (D) Western blotting was used to measure the levels of Cdc25A, p21, p53, and cyclin D1 in the normal chondrocyte cell line C-28/I2 (Control) as well as JJ012 and OUMS-27 cells. *p < 0.05 versus control.

Article Snippet: Transfection with 100 nM miR-497 mimic, antagomir, and negative control miRNA (RiboBio, Guangzhou China) or 1 μg CDc25A plasmid (NM_001789) (Origene, Rockville, MD, USA) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Molecular Oncology

Article Title: Integrative miRNOMe profiling reveals the miR ‐195‐5p– CHEK1 axis and its impact on luminal breast cancer outcomes

doi: 10.1002/1878-0261.70077

Figure Lengend Snippet: Relapse‐free survival probabilities for breast carcinoma patients stratified by subtype, therapy, and expression of studied genes. Relapse‐free survival (RFS) of adjuvant chemotherapy‐treated patients with luminal subtypes ( N = 171) (A) or solely the patients with the luminal A subtype ( N = 54) (B) stratified by the CHEK1 gene expression. RFS of adjuvant chemotherapy‐treated patients with luminal subtypes ( N = 171) (C) and solely the patients with the luminal B subtype ( N = 113) (D) stratified by the CCNE1 gene expression. Neoadjuvant chemotherapy‐treated patients with luminal subtypes ( N = 38) stratified by miR‐195‐5p (E) and CDC25A (F) gene expression levels. The optimal cut‐off for dividing patients with low vs. high expression was the 50th percentile (Q1‐Q2 vs Q3‐Q4) unless otherwise specified.

Article Snippet: We identified CHK1 (1:1000, mouse monoclonal antibody, 2G1D5, Novus Biologicals, Centennial, CO, USA), CCNE1 (1:200, mouse monoclonal antibody, CCNE1/2460, Novus Biologicals), CDC25A (1:500, mouse monoclonal antibody, #336445, R&D Systems, Minneapolis, MN, USA), ERα (1:000, rabbit monoclonal antibody, #8644, Cell Signaling, Danvers, Massachusetts, USA), cleaved caspase‐3 (1:500, rabbit monoclonal antibody, #9664, Cell Signaling), and cleaved PARP (1:750, rabbit polyclonal antibody, #9542, Cell Signaling).

Techniques: Expressing, Adjuvant, Gene Expression

Journal: Molecular Oncology

Article Title: Integrative miRNOMe profiling reveals the miR ‐195‐5p– CHEK1 axis and its impact on luminal breast cancer outcomes

doi: 10.1002/1878-0261.70077

Figure Lengend Snippet: Relative changes of the CHEK1, CDC25A, and CCNE1 gene expression in breast cancer cell lines transfected with miR‐195‐5p miRNA mimics compared to control in vitro . Baseline protein expression of the CHEK1, CDC25A, CCNE1, and ERα across BT‐20, MCF‐7, and T‐47D breast cancer cell lines is shown in (A). Relative changes in protein (B, C) and mRNA levels (D) in the same cell lines transfected with miR‐195‐5p miRNA mimics or negative controls for 24 h are presented. Cell lines treated with Lipofectamine RNAiMAX alone or with scrambled miRNAs were used as negative controls. The genes were considered significantly differentially expressed at the transcript level if the absolute value of fold change compared to control was ≥1.5 and P ‐value ≤0.05. The upper and lower lines of the boxplots represent the upper (75%) and lower quartile (25%), the line across the box represents the median, and the solid circles indicate outliers. Results of transcript expression measured by qPCR are presented as the mean ± SD of at least three independent experiments. Densitometric data of protein expression measured by western blot are presented as a percentage of the control (mean ± SD of three independent experiments), except for CDC25A in T‐47D cells and CCNE1 in BT‐20 cells (based on two replicates due to lack of expression in the third experiment). Western blot images show a representative experiment. Data were analyzed with an unpaired Student's t ‐test; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: We identified CHK1 (1:1000, mouse monoclonal antibody, 2G1D5, Novus Biologicals, Centennial, CO, USA), CCNE1 (1:200, mouse monoclonal antibody, CCNE1/2460, Novus Biologicals), CDC25A (1:500, mouse monoclonal antibody, #336445, R&D Systems, Minneapolis, MN, USA), ERα (1:000, rabbit monoclonal antibody, #8644, Cell Signaling, Danvers, Massachusetts, USA), cleaved caspase‐3 (1:500, rabbit monoclonal antibody, #9664, Cell Signaling), and cleaved PARP (1:750, rabbit polyclonal antibody, #9542, Cell Signaling).

Techniques: Gene Expression, Transfection, Control, In Vitro, Expressing, Western Blot

Journal: bioRxiv

Article Title: Non-Canonical Heme Oxygenase-1 Function in Hematopoietic Stem Cell Homeostasis and Aging

doi: 10.64898/2026.01.26.701757

Figure Lengend Snippet: (A) Annexin V assay results comparing the percentage of viable and apoptotic cells between HO-1 WT and HO-1 KO HSCs, and (B) HO-1 WT and HO-1 KO KLS cells. (C) Flow cytometry analysis of Chk1 expression and its phosphorylated form in HSCs, together with their ratio in both genotypes. (D) Flow cytometry analysis of Chk2 expression and its phosphorylated form in HSCs, together with their ratio in both genotypes. (E) Flow cytometry analysis of Cdc25a expression and its phosphorylated form in HSCs, together with their ratio in both genotypes. (F) Flow cytometry analysis of Cdk1 expression and its phosphorylated form in HSCs, together with their ratio in both genotypes. Points represent individual mice. Data are shown as mean ± SD.

Article Snippet: Following antibody clones were used for the intracellular staining: Cdc25A (clone F-6, Santa Cruz Biotechnology), Cdc25A Ser124 (Bioss Antibodies), Cdc25A Ser76 (Biorbyt), Cdc45L (clone JJo91-04, ThermoFisher Scientific).

Techniques: Annexin V Assay, Flow Cytometry, Expressing