Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: Cdc7 depletion in IMR90 fibroblasts causes cell cycle arrest in G1. (A) Time course of CDC7 mRNA knock-down (KD) in IMR90 cells relative to cells transfected with control-siRNA (CO). (B) Whole cell extracts (WCE) prepared from untreated (UT), CO and Cdc7KD cells were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (C) At the indicated time points, cell number was measured in UT, CO and Cdc7KD cell populations. (D) DNA content of UT, CO and Cdc7KD cells at 96 and 120 h post-transfection. (E) DNA content of double thymidine-arrested cells (DTB), CO and Cdc7KD cells 48 h after release from double thymidine block and transfection, and asynchronous Cdc7KD cells 48 h post-transfection. (F) At 96 h post-transfection, cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. DNA was stained with propidium iodide (PI). Numbers show the percentage of cells incorporating BrdU.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence containing four silent, single base pair mutations in the 21 bp CDC7-siRNA (oligo-A) interaction region was inserted into pCMV6-AC expression vector (OriGene) to fully abolish the siRNA effect.

Techniques: Transfection, Western Blot, Blocking Assay, Staining

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: CDK activity and replication initiation factors are downregulated in response to Cdc7 depletion. (A) WCE prepared from IMR90 cells transfected with control-siRNA (CO) and CDC7-siRNA (Cdc7KD) were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (B) WCE prepared from CO and Cdc7KD cell populations and from CO cells treated with 200 μM roscovitine (ROS) for 24 h (negative control) and MDA-MB231 breast cancer cells (positive control) were immunoprecipitated with anti-cyclin A and anti-cyclin E antibodies. Cdk2 immunoprecipitates (Cdk2 IP) were subjected to an in vitro kinase assay using recombinant truncated Rb protein (p56) as substrate. In vitro phosphorylation was detected with a specific anti-Rb-phospho-Thr-821 antibody. Note that lanes 1–8 were run on the same polyacrylamide gel and proteins transferred to the same PVDF membrane by semi-dry electroblotting. The membrane was subsequently cut as indicated for optimized immunodetection. (C) WCE and chromatin-bound protein fractions (CBF) prepared from untreated (UT), CO and Cdc7KD cells (96 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and histone H1—loading controls). (D) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) were analysed by western blotting with the indicated antibodies (β-actin—loading control). (E) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) and from cells treated for 24 h with 17 μM cisplatin (Pt) were analysed by western blotting with the indicated antibodies (β-actin—loading control).

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence containing four silent, single base pair mutations in the 21 bp CDC7-siRNA (oligo-A) interaction region was inserted into pCMV6-AC expression vector (OriGene) to fully abolish the siRNA effect.

Techniques: Activity Assay, Transfection, Western Blot, Negative Control, Positive Control, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Immunodetection

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: p53-dependent upregulation of Wnt/β-catenin signalling antagonist Dkk3 is required for Cdc7-depletion-induced cell cycle arrest. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) from untreated (UT), control-siRNA (CO) and CDC7-siRNA (Cdc7KD)-transfected IMR90 cells (72 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (B) At the same time point, CO and Cdc7KD cells were fixed and β-catenin, Myc and cyclin D1 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C, D) CF and NE samples prepared from CO, Cdc7KD and doubly depleted Cdc7/Dkk3 (Cdc7KD/Dkk3KD) cells 48 and 72 h post-transfection were analysed by immunoblotting with the indicated antibodies. (E) 72 h post-transfection CO, Cdc7KD and Cdc7KD/Dkk3KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X (inset: higher magnification) were detected by indirect immunofluorescence with anti-PCNA and anti-γH2A.X antibodies and a fluorescein-labelled secondary antibody. DNA was stained with propidium iodide (BrdU) or DAPI (PCNA and γH2A.X). Apoptotic cell death was detected in doubly depleted Cdc7KD/Dkk3KD cells by (F) phase contrast microscopy and by (G) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence containing four silent, single base pair mutations in the 21 bp CDC7-siRNA (oligo-A) interaction region was inserted into pCMV6-AC expression vector (OriGene) to fully abolish the siRNA effect.

Techniques: Transfection, Western Blot, Immunofluorescence, Staining, Microscopy

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: FoxO3a-mediated upregulation of ARF and CDK inhibitors is an essential step for arresting cell cycle progression in Cdc7-depleted cells. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) prepared from CO, FoxO3aKD, Cdc7KD and doubly depleted Cdc7/FoxO3a (Cdc7KD/FoxO3aKD) IMR90 cells 48 h post-transfection were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). Note that the faster migrating band represents the hypo-phosphorylated, active form of p27 and the slower migrating band its hyper-phosphorylated inactive form (Rodier et al, 2001; Chopra et al, 2002) (B) DNA content of Cdc7KD and Cdc7KD/FoxO3aKD cells 48 h post-transfection. (C) 48 h post-transfection CO, Cdc7KD and Cdc7KD/FoxO3aKD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody (inset: higher magnification). Chromatin-bound PCNA and γH2A.X were detected as described in the legend to Figure 3. Apoptotic cell death was detected 48 h post-transfection in doubly depleted Cdc7KD/FoxO3aKD cells (D) by phase contrast microscopy and (E) by immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence containing four silent, single base pair mutations in the 21 bp CDC7-siRNA (oligo-A) interaction region was inserted into pCMV6-AC expression vector (OriGene) to fully abolish the siRNA effect.

Techniques: Transfection, Western Blot, Microscopy

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: The Cdc7-depletion-induced cell cycle arrest is p15INK4B dependent. (A) Upregulation of p15 levels in Cdc7-depleted IMR90 cells was confirmed by immunoblotting WCE prepared from CO cells and Cdc7-depleted cells 48, 96 and 120 h post-transfection with antibodies against p15 and β-actin (loading control). (B) Cdc7KD and CO cells were fixed 96 h post-transfection and p15 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C) CF and NE prepared from CO, Cdc7KD and doubly depleted Cdc7/p15 (Cdc7KD/p15KD) cells 72 h post-transfection were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (D) 72 h post-transfection CO, Cdc7KD and Cdc7KD/p15KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X were detected as described in Figure 3 legend. Apoptotic cell death was detected 72 h post-transfection in doubly depleted Cdc7KD/p15KD cells by (E) phase contrast microscopy and by (F) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence containing four silent, single base pair mutations in the 21 bp CDC7-siRNA (oligo-A) interaction region was inserted into pCMV6-AC expression vector (OriGene) to fully abolish the siRNA effect.

Techniques: Western Blot, Transfection, Immunofluorescence, Microscopy

Journal: Viruses

Article Title: Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

doi: 10.3390/v12060582

Figure Lengend Snippet: Summary table of available protocols posted to the WHO’s website.

Article Snippet: US CDC, USA , N1 gene , 71 bp , LOD: 1x10 0.5 RNA copies/μL and 10 RNA copies/ μL for Qiagen EZ1 and Qiagen respectively. , Probe showed high sequence homology with SARS coronavirus and Bat Sars-like coronavirus , , , 20 μM primers, 5 μM probe; 15 μL total volume , For the RT-qPCR TaqPathTM 1- Step RT-qPCR Master Mix. For extraction, they recommend bioMérieux NucliSens ® systems, QIAamp ® kits, QIAGEN kits, Roche Kits and Invitrogen kits.

Techniques: Amplification, Concentration Assay, RNA Extraction, Sequencing, Diagnostic Assay, Positive Control, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: DNA Damage Induces G1 Arrest via p53-dependent Down-regulation of CDC7. (A) Schematic presentation of the synchronisation protocol (see Methods). p53 siRNA was introduced 12h prior and irradiation (IR) 8h after the end of serum starvation. Cells were collected for analysis 4h after release from the second Aphidicolin block. (B) Immunoblotting analysis of the nuclear extract (NE) from untreated (UT) and IR cells probed with the indicated antibodies. Orc4 was used as a loading control. (C) Immunoblotting analysis of whole cell extracts (WCE) from synchronised IMR90 cells treated with either IR (6 Gy) or with Doxorubicin (Doxo) independently or in conjunction with either CDC7 overexpression (CDC7 WT ) and/or p53 silencing (p53 KD ), probed with the indicated antibodies. β-actin was used as a loading control. (D) Immunoblotting analysis of nuclear extracts (NE) from synchronised IMR90 cells treated with either IR (6 Gy) or Doxo independently or in conjunction with either CDC7 overexpression (CDC7 WT ) and/or p53 silencing (p53 KD ), probed with the indicated antibodies. Orc4 was used as a loading control. (E) FACS analyses are shown as pie charts to demonstrate that the G1 arrest induced by IR or Doxo can be abrogated by either p53 knockdown (p53 KD ) or ectopic expression of CDC7 (CDC7 WT ), as demonstrated by the resumption of cell cycle progression into the S-phase.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Irradiation, Blocking Assay, Western Blot, Over Expression, Expressing

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: p53 Down-regulates CDC7 Transcript through miR-192/215 Pathway. (A) Immunoblotting analysis of whole cell extracts (WCE) and (B) bar diagram of cell populations with different DNA content as measured by FACS (values above the bars represent the percentage of cells with S-phase DNA content) from IMR90 cells demonstrate an inverse correlation between p53 and CDC7 protein levels during stress induced with a low dose of actinomycin D (ActD; 1 nM) in the presence or absence of p53 (via knockdown with siRNA). (B) Representative of 3 independent experiments. (C) qRT-PCR quantification of relative CDC7 mRNA expression levels (-fold change) in cells treated with either ActD or DMSO, or with p53 siRNA plus control (non-targeting) siRNA NT compared with siRNA NT treatment alone (CTRL). p53 silencing combined with ActD treatment was compared to cells treated with ActD only. (C) Mean ± SEM from 3 independent experiments (D) IMR90 cells were transfected with either p53 siRNA for 48h or CDC7 expressing plasmid (CDC7 WT ), miR-192/215 siRNA alone or miR-192/215 siRNA together with miR-192/215 synthetic mimics (miR-Mimics) for 24h and together with a low dose of ActD (1 nM). Results were compared to either non-targeting control siRNA plus DMSO (CTRL) or ActD treated cells (ActD). Values above the CDC7 bands indicate CDC7 mRNA levels as measured by qRT-PCR relative to the ActD treatment (lanes 1–6) and the values above the p53 bands indicate the residual p53 mRNA after p53 silencing relative to the ActD treatment as measured by qRT-PCR (lanes 2 and 3). (E) DNA content distribution analysis of samples treated as described in (D) is presented as a bar diagram. Values above the bars indicate the percentage of S-phase cells. Representative of 3 independent experiments.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

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

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: Stress-induced Proteolysis of CDC7 is Dependent on CDC4-type Phospho-degrons (CPDs). (A) IMR90 cells were treated with cycloheximide (CHX, 10 μg/ml) in the presence of either DMSO (CTRL) or Actinomycin D (ActD; 1nM). Cells were harvested at the indicated time points and whole cell extracts (WCE) were analyzed by immunoblotting with an antibody against CDC7. (B) Immunoblotting analysis of WCE prepared from IMR90 cells that were first treated with either p53 siRNAs (48h) or miR-192/215 siRNA (24h) and then treated with ActD in the presence or absence of MG-132 (10 μM). (C) The putative Fbxw7 recognition motifs (CPDs) in the CDC7 protein sequence. The CDC4-phospho degron (CPD) consensus sequence is xT/SPxxT/S/x, where x is any amino acid residue. (D) Alignment of the phosphodegron motifs (CPDs) present in known Fbxw7 substrates with 5 CPDs in the CDC7 primary protein sequence. Positions of the key amino acid residues are highlighted in blue. (E) CDC7 structure (PDB 4f9a) shown as a ribbon with indicated positions of CPDs. Positions of CPDs 1, 2, 4 and 5 were predicted by modeling using iTasser and Phyre2 (see Methods and Fig. S4); Thr 376 in CPD3 is shown in red, ATP is shown in magenta and the Mg atom is shown as a yellow sphere. (F) CDC7 mutated on CPDs (CPDs 3, 4 and 5, CDC7 CPD ) and ectopically-expressed for 48h in IMR90 cells demonstrated more pronounced resistance to degradation compared to CDC7 WT in the absence or presence of ActD (1 nM). CTRL represents empty vector and DMSO treatment; β-actin was used as a loading control. (G) DNA content distribution analysis of samples analyzed in (F) shown as a bar diagram. Values above the bars indicate the percentage of cells with S-phase DNA content. (G) Representative of 3 independent experiments.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Western Blot, Sequencing, Plasmid Preparation

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: Stress-induced CDC7 Proteolysis is Mediated by Fbxw7β and GSK3β kinase. (A) Immunoblotting analysis of whole cell extracts (WCE) from IMR90 cells after treatment with Actinomycin D (ActD; 1 nM, 24h) in the presence or absence of p53 siRNA. Samples probed with anti-Fbxw7β antibody (Fbxw7β*) were prepared from cytoplasmic fractions. (B) Immunoblotting analysis of WCE with the indicated antibodies demonstrates CDC7 stabilization after Fbxw7β silencing (Fbxw7β K D ) for 48h in control (CTRL) IMR90 cells or IMR90 cells treated with ActD for 24h. CTRL represents non-targeting siRNA and DMSO treatment. (C) DNA content distribution profiles for the indicated treatments as described in (B) presented as a bar diagram. Values above the bars indicate the percentage of cells with S-phase DNA content. (C) Representative of 3 independent experiments. (D and E) Immunoblotting analysis of WCE after CDC7 or ubiquitin pull-down in (d) IMR90 or (e) HCT116 Fbxw7 +/+ and Fbxw7 −/− cells in the presence or absence of ActD (1nM, 24h with DMSO as a negative control) and the proteasome inhibitor MG-132 (10 and 20 μM for IMR90 and HCT116, respectively) where indicated. Ubiquitinated CDC7 is revealed in presence or absence of stress (ActD) only after inhibition of the proteasome MG-132. The intense cut band on the bottom are immunoblobulins (Ig) (F) Immunoblotting analysis of the in vitro kinase assay reactions performed using the recombinant active CDC7/ASK kinase complex in the presence or absence of recombinant active GSK3β and the presence or absence of ATP. The reaction samples were analyzed with 2 different anti-phospho-threonine antibodies (pThr Ab1 and pThr Ab2), anti-pThr376 (CDC7 pT376) and an antibody recognizing all phosphorylated serines (pSer). Recombinant protein loading was controlled with anti-CDC7 and anti-GSK3 antibodies and antibodies specifically detecting the GST and His tags.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Western Blot, Negative Control, Inhibition, In Vitro, Kinase Assay, Recombinant

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: Post-transcriptional and Post-translational p53-dependend Pathways Cooperate to Down-regulate CDC7 in Cells Undergoing DNA Damage. (A) Immunoblotting analysis of whole cell extracts (WCE) from IMR90 cells treated with either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA, GSK3β siRNA or Cdh1 siRNA. β-actin was used as a loading control. (B) DNA content distribution analysis of indicated samples as described in (A) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (B) Representative of 3 independent experiments. (C) Residual p53-, Fbxw7β-, miR-192/215- and GSK3β mRNA as measured by qRT-PCR in percentages (%) relative to the respective control (CTRL). (C) Mean ± SEM from 3 independent experiments (D) 2 μM Doxorubicin (Doxo) was added to IMR90 cells for 48h, after 24h pre-treatment with either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA or GSK3β siRNA. CTRL represents the combination of non-targeting control siRNA and DMSO treatment. (E) DNA content distribution analysis of indicated samples as described in (D) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (E) Representative of 3 independent experiments. (F) 1 nM Actinomycin D (ActD) was added to HCT116 cells for 24h, after 24h pre-treatment with following siRNAs: either p53 siRNA, miR-192/215 siRNA, Fbxw7β shRNA, a combination of miR-192/215 siRNA and Fbxw7β shRNA or GSK3β siRNA. CTRL represents the combination of non-targeting control siRNA and DMSO treatment. (G) DNA content distribution analysis of indicated samples as described in (F) is presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (G) Representative of 3 independent experiments.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Western Blot, shRNA, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: p21 complements the CDC7 downregulation under stress. (A) Levels of CDC7 and its downstream target MCM2 (phospho-Ser) pS108 protein according to immunoblotting analysis of WCE after silencing p53 (p53 KD ) for 48h compared to the combination of miR-192/215 silencing and/or p21 silencing in Doxorubicin (Doxo) treated IMR90 cells. (B) Efficiency of p21 silencing (%) in cells treated with Doxo or Doxo and miR-192/215 siRNA as measured by qRT-PCR. (C) Mean ± SEM from 3 independent experiments (C) DNA content distribution profiles of indicated treatments in (A) presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (C) Representative of 3 independent experiments. (D) Immunoblotting analysis of the WCE from HCT116 p21 −/− and p21 +/+ cells in the presence or absence of Actinomycin D (ActD; 1 nM, 24h, DMSO as a negative control) and CDK2 silencing (CDK2 KD ) shows a reduction of CDC7 levels in p21 −/− cells after CDK2- and CDC7 phospho-Thr376 (CDC7 pT376) inhibition. (E) DNA content distribution profiles of samples described in (D) presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (E) Representative of 3 independent experiments. (F) Effect of CDK2 inhibition by Roscovitine (Rosco, 40μM) on CDC7 recovery after p21 silencing in ActD-treated IMR90 cells as detected by immunoblotting. Phospho-Thr376 (pT376) CDC7 antibody was used to assess CDK2 inhibition by Rosco. (G) Immunofluorescence analysis of CDC7 levels in HCT116 p21 −/− cells treated with either DMSO control, ActD (1 nM) or ActD (1 nM) and p21 siRNA in the presence or absence of Rosco (40 μM). Actin immunostaining (FITC) was used to visualize the cytoplasm.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Western Blot, Quantitative RT-PCR, Negative Control, Inhibition, Immunofluorescence, Immunostaining

Journal: Cell Cycle

Article Title: p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

doi: 10.1080/15384101.2016.1231281

Figure Lengend Snippet: Sustained overexpression of active CDC7 down-regulates p53, abrogates S-phase entry control and leads to DNA damage - biological implications of the p53-CDC7-regulatory pathway. Whole cell extracts (WCE) from IMR90 cells transfected every 48h with either wild type- (CDC7 WT ) in (A) or the kinase-inactive CDC7 (CDC7 KAD ) mutant in (B) over a 20 day time course and immunoblotting analysis for CDC7, p53 and γH2A.X levels (DNA damage marker). Cells transfected with an empty vector control (CTRL) acted as a negative control in both (A) and (B). β-actin was used as a loading control throughout. (C) DNA content distribution profiles of indicated treatments in (A) presented as a bar diagram. Values above the bars represent the percentage of cells with S-phase DNA content. (C) Representative of 3 independent experiments. (D) Schematic representation of the p53-dependent pathways responsible for controlling G1/S transition. The novel pathway is shown in blue. Genotoxic stress (irradiation, DNA damage and cytotoxic drugs) stabilises and activates the p53 tumor suppressor. p53 then transcriptionally up-regulates p21, which inhibits CDK2 activity and consequently prevents the CDK2-dependent phosphorylation of CDC7 at Thr376 located in CDC4-phosphodegron 3 (CPD3). Likewise, phosphorylation of Ser54, a CDK2 target in CDC6, is prevented, marking it for degradation by the APC/Cdh1 complex. Lack of CDC7 Thr376 phosphorylation facilitates CDC7 phosphorylation by stress-induced GSK3β on the other CPDs, and leads to the subsequent interaction with Fbxw7β E3 ubiquitin ligase and degradation. p53 induces both miR-192/215 which provides CDC7 transcript down-regulation, and Fbxw7β which targets CDC7 protein for degradation in response to genotoxic stress. CDC7 exerts negative feedback onto p53 through its kinase activity. The feedback loop allows CDC7 to retain its high levels and ensures cell entry into S-phase. (E) Stress-related CDC7-dependent cell cycle checkpoints. Genotoxic stress triggers (1) p53-dependent downregulation of CDC7 and (2) activation of a p53-dependent DNA origin activation checkpoint (OAC) inducing a robust G1/S cell cycle arrest. Increased CDC7 activity can down-regulate p53 levels via a negative feedback loop (3) and override the G1/S checkpoint. During S phase (4) in response to replication stress, activation of the ATR/Chk1 checkpoint results in stabilization of CDC7 that prevents replication fork collapse (40, 41). DNA-RI - DNA replication initiation.

Article Snippet: For rescue experiments, the full 1725 bp CDC7 cDNA sequence inserted into pCMV6-AC expression vector (OriGene) was used, as described in.

Techniques: Over Expression, Transfection, Mutagenesis, Western Blot, Marker, Plasmid Preparation, Negative Control, Irradiation, Activity Assay, Activation Assay

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Detection of Proteasome 26S subunit, non-ATPase 3 (PSMD3) expression level in normal and malignant breast cancer (BC) cell lines and human BC tissues. ( A ) PSMD3 expression at protein level in Human epidermal growth factor receptor 2 (HER2) positive versus HER2 negative were detected by Western blot; normal (MCF10A) and cancerous human breast cell lines (Au565, BT-474, SKBR3, HCC-1419, HCC-1954, MDA-MB231, MDA-MB 468, MDA-MB 436, B-20, BT-549, H578T). β-actin served as internal control. PSMD3 bands densities were normalize to β-actin. ( B ) Detection of PSMD3 expression at mRNA level in 15 pairs normal versus tumor breast tissue samples were detected by reverse transcriptase (RT)- polymerase chain reaction (PCR). β-actin served as internal control. ( C ) The PSMD3 mRNA expression profiles of paired human breast tumor (red lines) and normal (green lines) tissues ( n = 176) were detected by real-time PCR. ( D ) Comparison of PSMD3 mRNA expression between normal (N) and tumor (T) pairs ( n = 176), copy number (mg /mRNA). PSMD3 mRNA expression levels in 23 patients’ samples in which PSMD3 mRNA expression was higher in normal tissue than in tumor tissue (Group 1) versus 153 patient samples in which expression was higher in tumor tissue than normal tissue (Group 2). Error bars indicate the 95% confidence interval. Data was analyzed with 2-sided paired t -test (*** p <0.001). ( E ) Analysis of PSMD3 copy number levels between BC subtypes, including (Estrogen Receptor; ER− vs. ER−, Progesterone receptor; PR− vs. PR+, HER2− vs. HER2+, and Triple Negative Breast Cancer; TNBC vs. Non-TNBC). Error bars indicate the 95% confidence interval. Data was analyzed with independent t-test (* p < 0.05). ( F ) Representative images for PSMD3 Immunohistochemistry (IHC) immunostaining Tumor area versus normal area (Upper panel) (100× and 400×) and hematoxylin and eosin staining (lower panel) (100× and 400×).

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Expressing, Western Blot, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunohistochemistry, Immunostaining, Staining

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Direct interaction between HER2 and PSMD3 in BT474, SKBR3, and HCC-1419. ( A ) Total cell lysates from HER2+ cells were incubated with either PSMD3 or HER2 antibodies at 4 °C overnight. Immunoprecipitation (IP) were subjected to western blot analysis with anti-PSMD3 or anti-HER2 antibodies. ( B ) BT-474, SKBR3, HCC-1419, and MDA-MB231 were stained with PSMD3 and HER2 antibodies at 4 °C overnight. Secondary antibodies conjugated with Rhodamine and FITC against anti-PSMD3 and anti-HER2, respectively, were incubated for an hour at room temperature. Confocal Fluorescence resonance energy transfer (FRET) assay was used to analyze the protein–protein interaction. Scale Bar is 25 µM.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Incubation, Immunoprecipitation, Western Blot, Staining, Fluorescence, Förster Resonance Energy Transfer

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: PSMD3 expression is essential to maintain HER2 stability. ( A ) Twenty-four hours after transfection of BT474, SKBR3, and HCC-1419 with either scramble (Sc), PSMD3-Si001, or PSMD3-Si002. The cell lysates were collected and analyzed by western blot to detect the PSMD3 and HER2 protein levels. ( B ) Scramble, PSMD3-si001, or PSMD3-Si002 for BT474 and SKBR3 were treated with Cycloheximide (CHX) at 20 µg. The cells lysates were obtained at the indicated time points and analyzed by western blot. ( C ) BT474, SKBR3, and HCC-1419 were treated with Scramble, PSMD3-Si001, or PSMD3-Si002 for 24 h and the cells were lysed. The samples were resolved by SDS-PAGE and subjected to western blot analyses with indicated antibodies. ( D ) BT474, SKBR3 and HCC-1419 were transfected with Scramble, PSMD3-Si001or PSMD3-Si002 for 24 h, then the cells were lysed and the proteins were extracted. The samples were analyzed by western blot to detect the PSMD3, HER2, T-AKT, p-AKT, T-ERK and p-ERK, β-actin served as internal control.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Expressing, Transfection, Western Blot, SDS Page

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Silencing of PSMD3 inhibits cell proliferation and induces cell apoptosis. ( A ) Cell proliferation assay for BT474 and SKBR3. After transfection the cells with Scramble (Sc), PSMD3-Si001, or PSMD3-Si002, cells (2X10 4 ) were seeded in a 24-well dish and cells number were counted at day 1, day 2, and day 4. Data are presented as the mean ± S.D * p < 0.05. ( B ) BT474 and SKBR3 were seeded in a 12-well culture plate after being treated with the indicated plasmids. After 10 days, the cells were fixed and stained with crystal violet. Data are presented as the mean ± S.D (* p < 0.05 *** p < 0.001). ( C ) Annexin V, cell apoptosis assay by flow cytometry. Twenty-four hours after treating the cells with the indicated plasmids, cells were collected and washed two times with PBS and stained with PE-Annexin or 7AAD. Data are presented as mean ± S.D (* p < 0.05 *** p < 0.001). ( D ) The expression of cell cycle and apoptotic related markers for the HER2 positive cell lines were detected by western blot (PSMD3, HER2, CDK4, CDK6, PARP, and caspase3). β-actin served as internal control.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Proliferation Assay, Transfection, Staining, Apoptosis Assay, Flow Cytometry, Expressing, Western Blot

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Enhancing the ubiquitination and lysosomal degradation pathway through silencing of PSMD3. ( A ) BT-474 and HCC-1419 control. Scramble and PSMD3-Si001 were co-transfected with or without ubiquitin plasmid for 24 h and then lysed. The cell lysates were incubated with antibodies against Ubiquitin (HA), HER2, and PSMD3. β-actin served as a control. ( B ) BT-474 was treated with Scramble and PSMD3-Si001 were co-transfected with or without ubiquitin plasmid for 24 h and then treated with or without MG132 at a concentration of 5µM for 8 hours and lysed. The cell lysate was incubated with the indicated antibodies. ( C ) Confocal microscope pictures of SKBR3 and BT474 treated with scramble or PSMD3 for 24 h and incubated with HER2 and LAMP-1 lysosomal marker antibodies and showing HER2-LAMP-1 co-localization. Scale bar is 25 µM. ( D ) B-474, SKBR3 cell lysates were incubated with PSMD3 and USP14 antibodies. Western blot analysis to detect PSMD3 and USP14 protein levels was used. β-actin served as a control. ( E ) B-474 transfected with Scramble, PSMD3 si001, or PSMD3 si002 for 24 h. Control cells were treated with Wortmanine inhibitor for 3 hours. Western blot analysis to detect PSMD3, HER2, p-AKT, and USP14 protein levels was used. β-actin served as a control. ( F ) Overexpression of PSMD3 in BT474, HS578T, and BT459. Western blot data showing the expression of PSMD3, HER2, and β-actin; * indicates the PSMD3 conjugated with Yellow fluorescent protein (YFP) fusion protein, where the band shifted upward.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Transfection, Plasmid Preparation, Incubation, Concentration Assay, Microscopy, Marker, Western Blot, Over Expression, Expressing

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Detection of PSMD3 expression by IHC staining and association of PSMD3 with clinical overall survival. ( A ) Representative images for PSMD3 IHC scoring system. The scoring system was determined as Negative (0), Weak (1+), Moderate (2+), and Strong (3+) based on PSMD3 staining intensity. Scale bar is 1 µM (10×). ( B ) Representative images for low H-score (normal breast cells) versus high H-Score (tumor breast cells). Note the cytoplasmic and nuclear staining in tumor cells (original magnification 100× and ×400). ( C ) Histological analysis of PSMD3 expression in BC patients based on H-score value ( p < 0.001 ***). ( D ) Kaplan–Meier survival curves of breast cancer patients. A comparison of low- and high-PSMD3 H-score in HER2+ versus total cases. The difference between the survival curves was calculated using the log-rank test. PSMD3 expression level was calculated using PSMD3 protein H-score. ( E ) Kaplan–Meier Plotter survival curves of breast cancer (BC) patients. Comparison of low- and high-PSMD3 mRNA level in BC Overall survival (OS), Relapse-free survival (RFS), and progression-free survival (PFS) and in HER2+ BC; (OS) and distant metastasis-free survival (DMFS). The difference between the survival curves was calculated using the log-rank test.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: Correlation between PSMD3 gene expression and Clinicopathological parameters of breast cancer using TCGA data ( n = 1212).

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Expressing

Journal: Cancers

Article Title: Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation

doi: 10.3390/cancers11040527

Figure Lengend Snippet: A schematic molecular model for PSMD3 in the stabilization of HER2. USP14 is a DUB enzyme that trims ubiquitin (Ub) from substrates. PSMD3 interacts with HER2, which maintains HER2 and HER2 signaling pathways. Loss of PSMD3 function led to destabilization of HER2 and enhanced ubiquitination and association with decreased HER2 level and lysosomal degradation pathway. The major HER2 signaling pathways (AKT and ERK) were inhibited, leading to the inhibition of cell proliferation and the induction of cellular apoptosis.

Article Snippet: For PSMD3 overexpression, a vector that contained the PSMD3 gene (full-length CDC mRNA 1605 bp) was purchased from Applied Biological Materials (ABM) Inc. (British columbia, Canada).

Techniques: Inhibition