Journal: The Prostate

Article Title: Adaptive auto-regulation of androgen receptor provides a paradigm shifting rationale for bipolar androgen therapy (BAT) for castrate resistant human prostate cancer.

doi: 10.1002/pros.22504

Figure Lengend Snippet: Fig. 1. AR and RC protein expression in human prostate cancer cell lines and antibody validation for IP and IB analysis. A: IB of AR protein in AR-expressing LNCaP, CWR22Rv1, LAPC4, and VCaP cells compared to AR-non expressing PC-3 prostate cancer cells using an N-terminal specific anti-AR antibody. Nuclear protein extracts from 105 cells were loaded per well for each of the lines. B: IB of AR in cytosolic (denoted cyto. extract) versus nuclear (denoted nucl extract) extract from105 LNCaP cells.C: IB of Orc1, Orc2, Cdc6, Cdt1, and Mcm2 in nuclear extracts from 105 LNCaP cells.D:IB for indicatedproteinincytosolic (denotedcyto extract) versus nuclear (denoted nucl extract) extracts and IB of IP using specific antibodyagainst theindicatedprotein(denotedIP)orusing non-specific IgG (denoted IP:IgG) from nuclear extracts of LAPC4 and LNCaP cells.E: IB of IP using non-specific IgG (denoted IP:IgG) versus specific antibody against the indicated protein (denoted IP) versus flow through from the specific IP (denoted IP:FT) from nuclearextractsofLNCaPandPC-3cells.

Article Snippet: The antibodies used for IB detection of: AR (mouse monoclonal 441sc-7305 from Santa Cruz); Orc1 (rat monoclonal 7A7 sc-23887, Santa Cruz); Orc2 (rat monoclonal 3G6 cat# 4736 from Cell Signaling); Cdc6 (mouse monoclonal 180.2 sc-9964 from Santa Cruz); Cdt1 (rabbit polyclonal H-300 sc-28262 from Santa Cruz), and Mcm2 (mouse monoclonal cat # 610700 num; 610700 from BD Pharmingen).

Techniques: Expressing, Biomarker Discovery

Journal: The Prostate

Article Title: Adaptive auto-regulation of androgen receptor provides a paradigm shifting rationale for bipolar androgen therapy (BAT) for castrate resistant human prostate cancer.

doi: 10.1002/pros.22504

Figure Lengend Snippet: Fig. 7. AR binds with RCs in early-G1 in AR-dependent castra- tion-resistantprostate cancer cells. A:Temporal change in expres- sion detectedby IB of Orc2,Cdt1,Mcm2, AR,Orc1,Cyclin D1,Cdk4, Cdc6, and Cdk2 in LNCaP cells progressing through cycle after release from early-G1 arrest. Protein lysates from 105 cells were loadedperlane.Expressionin asynchronouslygrowing LNCaPcells (denoted Asynchro). B: IB of AR from nuclear co-IP using non- specific IgG (denoted IgG) versus specific antibody to either Orc2 or Cdc6 in asynchronously growing LNCaP cells (denoted control) versus cells arrestedin early-G1 (denoted 0 hr) or 8, or 24 hr post- release.C:IB ofARandORC2fromnuclearco-IPusingnon-specific IgG (denoted IgG) versus specific antibody to Orc2 in asynchro- nously growing LNCaP cells (denoted bicalutamide) versus cells treated for 4 days with 10 mM bicalutamide (denoted bicalutamideþ). D: IB of AR and ORC2 from nuclear extracts of bicalutamide untreated (denoted) versus treated (denotedþ) from (C). E: IB of AR and ORC2 from nuclear co-IP using specific antibody to Orc2 on LNCaP cells treated for 4 days with 10 mM bicalutamide.

Article Snippet: The antibodies used for IB detection of: AR (mouse monoclonal 441sc-7305 from Santa Cruz); Orc1 (rat monoclonal 7A7 sc-23887, Santa Cruz); Orc2 (rat monoclonal 3G6 cat# 4736 from Cell Signaling); Cdc6 (mouse monoclonal 180.2 sc-9964 from Santa Cruz); Cdt1 (rabbit polyclonal H-300 sc-28262 from Santa Cruz), and Mcm2 (mouse monoclonal cat # 610700 num; 610700 from BD Pharmingen).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Oncotarget

Article Title: RACK1 promotes lung cancer cell growth via an MCM7/RACK1/Akt signaling complex

doi: 10.18632/oncotarget.17120

Figure Lengend Snippet: ( A ) A549 cells were transfected with siRACK1, siCon, GFP-RACK1, or GFP. MCM7 immunoprecipitates were probed for P-Ser, P-Thr, and MCM7 as indicated. ( B ) A549 cells were transfected with siRACK1 or siCon. MCM7 immunoprecipitates were probed for Cdt1, MCM4, MCM6, and MCM7. ( C ) A549 cells were treated with siRACK1, siCon, GFP-RACK1, or GFP as indicated. The chromatin (Chr) and non-chromatin (Non-Chr) fractions of these cells were purified and immunoblotted with anti-MCM7 antibodies. Antibodies against Histone 3 and GADPH were used as internal controls.

Article Snippet: Antibodies for MCM4, MCM6, Cdt1, p27, E2F1, and Histone 3 were from Proteintech Group.

Techniques: Transfection, Purification

Journal: Clinical Cancer Research

Article Title: The NEDD8-Activating Enzyme Inhibitor MLN4924 Disrupts Nucleotide Metabolism and Augments the Efficacy of Cytarabine

doi: 10.1158/1078-0432.ccr-14-1960

Figure Lengend Snippet: Figure 3. CDT1 stabilization is not required for MLN4924 to augment ara-C activity. A, knockdown of CDT1. Lentiviral shRNA was utilized to knock down CDT1 expression. MOLM-13 cells infected with CDT1-directed or nontargeted control shRNA were treated with 100 nmol/L MLN4924, 100 nmol/L ara-C, or both drugs for 24 hours. Knockdown efficiency was evaluated by immunoblotting. Tubulin documented equal protein loading. B, CDT1 impairment does not significantly impact the proapoptotic effects of the MLN4924/ara-C combination. Cells infected with nontargeted control or CDT1-directed shRNA were treated with 100 nmol/L MLN4924, 100 nmol/L ara-C, or both drugs for 48 hours. Drug-induced apoptosis was quantified by PI/FACS analysis. n ¼ 3 SD.

Article Snippet: Cells were imaged using fluorescent microscopy and tail moments (product of DNA amount in tail and distance of tail migration) from 50 cells per slide were calculated. shRNA knockdown of CDT1 MOLM-13 human AML cells were infected with lentiviral particles containing nontargeted (control) or targeted shRNA directed at CDT1 (Santa Cruz Biotechnology) according to the manufacturer's instructions.

Techniques: Activity Assay, Knockdown, shRNA, Expressing, Infection, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: Functional analysis of Cdt1 variants by rereplication induction. (A) Illustration of the relative location and amino acid substitution of the alleles chosen in this study; polyhistidine and HA epitope tags and relevant binding domains are also marked. (B) Immunoblot of inducible expression of stably integrated HA-tagged WT Cdt1 in U2OS cells. Cells were grown in 0, 0.05, 0.1, and 1 µg/ml doxycycline (dox), respectively. (C) Analytical flow cytometry profiles of U2OS cells expressing vector, ectopic HA-tagged WT Cdt1, or the indicated HA-tagged Cdt1 variants. Cells were treated with 1 µg/ml dox for 72 h and pulse-labeled with EdU for 30 min before being harvested. An illustration of the gating scheme is also shown; “>4C DNA” are cells that have undergone DNA rereplication. (D) Immunoblots of Cdt1 expression from C. Light Exp., light exposure; Dark Exp., dark exposure. (E) The percentage of cells with >4C DNA content in at least four biological replicates. Bars represent mean and SD. **** p value < 0.0001; ** p value < 0.005; * p value < 0.05; n.s. = not significantly different.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Functional Assay, Binding Assay, Western Blot, Expressing, Stable Transfection, Flow Cytometry, Plasmid Preparation, Labeling

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: DNA damage and cell proliferation defects from Cdt1 variant overproduction. (A) Immunoblots of HA-tagged Cdt1 (anti-HA antibody), pChk1 (S345), and total Chk1 in U2OS cells grown in 1 µg/ml dox for 48 h. (B) Graph of pChk1 (S345) induction normalized to WT Cdt1. Bars represent mean and SD of three biological replicates. **** p value < 0.0001; *** p value = 0.0001; * p value < 0.05; n.s. = not significantly different. (C) Top, Representative vector and WT Cdt1 control colony-forming assays. Cells were plated at low density in the presence or absence of 1 µg/ml doxycycline (dox) and grown for ∼10 d. Bottom, A technical replicate plate was harvested after 72 h to assay for ectopic Cdt1 expression by immunoblotting with anti-Cdt1 antibody. (D) Relative colony formation normalized within each experiment to the vector control; values represent at least three biological replicates. Bars represent mean and SD. ** p value < 0.005; n.s. = not significantly different.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Variant Assay, Western Blot, Plasmid Preparation, Control, Expressing

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: Functional analysis of MCM loading. (A) Analytical flow cytometry profiles of chromatin-bound MCM in U2OS cells treated with 100 nM control siRNA (left) or Cdt1 siRNA (right). Cells were pulse-labeled with 10 µM EdU for 30 min before harvesting and extraction of soluble MCM. Bound (unextracted) MCM was detected with anti-MCM2 antibody, and cells were stained with DAPI for total DNA content. Blue: MCM Bound positive, EdU negative, G1 DNA content; orange: EdU positive, MCM Bound positive; gray: EdU negative, MCM Bound negative. (B) Histograms of the G1 MCM Bound positive, EdU negative (i.e., blue in A) cells from both samples in A. Bound MCM on the x -axis and normalized cell counts on the y -axis (counts normalized to siControl). (C) Histograms of G1 MCM Bound positive cells depleted of endogenous Cdt1 and expressing each Cdt1 variant compared with WT Cdt1 as in B. siRNA transfected cells were cultured in 0.002–0.006 µg/ml doxycycline for 72 h before EdU labeling and processing as in A. (D) Immunoblot of endogenous and ectopic Cdt1 from C detected with anti-Cdt1 antibody. (E) Complementation of G1(green/blue) and early S (orange) MCM loading normalized to WT Cdt1. Mean MCM Bound loading intensity of each variant was divided by the mean MCM loading intensity of WT Cdt1 within each experiment. Early S phase is defined as G1 DNA content and EdU-positive indicated by the bracket in A; see also Supplemental Figure S3B. Bars represent mean and SD of three biological replicates. * p value < 0.05; ** p value < 0.005 where indicated; otherwise the difference between WT Cdt1 and variant was not significant.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Functional Assay, Flow Cytometry, Control, Labeling, Extraction, Staining, Expressing, Variant Assay, Transfection, Cell Culture, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: Relative MCM binding. (A–D) WT and the indicated Cdt1 variants were transiently expressed in HEK 293T cells and immunoprecipitated using the HA epitope tag. Portions (2%) of whole-cell lysates and bound proteins were probed for HA-Cdt1 (anti-HA antibody) and for MCM2 as a marker of the MCM complex; immunoglobulin G or beads were used as controls (CTRL). (E) Top, Homology model of the human MCM2-7-Cdt1 complex. The yeast OCCM structure (PDB ID: 5UDB) was used as a template to model the human MCM2-7 complex; numbers indicate individual MCM subunits, and colors are similar to those in . The structures of the human C-terminal Cdt1 winged helix, “Cdt1-CTD” (PDB ID: 2WVR), and the mouse Cdt1 central/middle domain, “Cdt1-MD” (PDB ID: 3A4C), were used to model hCdt1–hMCM interactions. Bottom, left, Magnified view of the proposed interacting surfaces with R210 highlighted in green. Bottom, right, Magnified view of the proposed interacting surfaces with R462 and E468 highlighted in green.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Binding Assay, Immunoprecipitation, Marker

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: Cdt1-A66T impairs cyclin A and Skp2 binding but does not stabilize Cdt1 in S phase. (A) Illustration of SCF Skp2 -dependent degradation of WT Cdt1 via CDK-mediated phosphorylation at threonine 29. (B) Cells were cultured in 1 µg/ml dox (high) for 18 h, lysed, and incubated with nickel–agarose to retrieve His-tagged Cdt1. Portions of whole cell lysates (2%) and bound complexes were probed for the indicated proteins (bottom panel anti-HA antibody). (C) Top, Illustration of Cdt1 degradation and accumulation during the cell cycle. Bottom, U2OS cells expressing Cdt1-WT and Cdt1-A66T were synchronized by double-thymidine/nocodazole (lanes 1–5) or double-thymidine (lanes 6–10) block and released into fresh medium. Time points were taken after release and analyzed by immunoblotting with anti-Cdt1 antibody. A nonspecific band serves as a loading control. (D) The intensity of WT or A66T Cdt1-Venus expressed in U2OS cells imaged during asynchronous proliferation every 10 min. Traces are the average Venus intensity in arbitrary units from mitosis (0%) to mitosis (100% cell-cycle progression); n = 50 cells. White circles denote the beginning and end of the S phase as determined by the localization of stably coexpressed fluorescently tagged PCNA. (E) Heat map of fluorescence intensity of Cdt1 WT-Venus (left) and Cdt1-A66T- Venus (right) in 50 randomly selected U2OS cells. Maps from individual cells are arranged according to the duration of the cell cycles; colors indicate differences in fluorescence levels. White dots in each track denote the beginning and end of the S phase as determined by the localization of stably coexpressed fluorescently tagged PCNA.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Binding Assay, Phospho-proteomics, Cell Culture, Incubation, Expressing, Blocking Assay, Western Blot, Control, Stable Transfection, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: CDK-Cdt1 binding suppresses rereplication independent of the Cdt1 phosphodegron. (A) Analytical flow cytometry profiles of cells treated with 1 µg/ml doxycycline for 48 h and analyzed as in . Cy: cyclin/CDK binding motif mutant; 2A: Cdt1 T29A, S31A. (B) The percentage of cells with >4C DNA content in at least three biological replicates. Bars represent mean and SD. * p value < 0.05; n.s. = not significantly different. (C) HA-tagged Cdt1 was detected by immunoblotting whole-cell lysates from A with anti-Cdt1 antibody; a nonspecific band serves as a loading control; Vec. = Vector.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Binding Assay, Flow Cytometry, Mutagenesis, Western Blot, Control, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

doi: 10.1091/mbc.E18-04-0242

Figure Lengend Snippet: Model of cell proliferation defects from both hypomorphic or hypermporphic Cdt1 variants. (A) WT Cdt1 supports normal MCM loading/origin licensing and normal DNA replication in the S phase. (B) The A66T variant is impaired for CDK-mediated repression, resulting in relicensing and rereplication. The R210C, R462Q, and E468K variants are impaired for MCM2-7 binding in the G1 phase, leading to slow origin licensing and thus to delayed G1 progression. Both scenarios ultimately lead to proliferation defects.

Article Snippet: To generate stable isogenic cell lines, U2OS cells were cotransfected with flippase recombinase (Flp) and a Cdt1 expression vector derived from pcDNA5/FRT/TO-Venus-Flag-Gateway (1124), a gift from Jonathon Pines (The Institute of Cancer Research, London) (Addgene plasmid # 40999), using X-tremeGENE HP DNA transfection reagent (Roche).

Techniques: Variant Assay, Binding Assay