Journal: Pharmaceutics

Article Title: Maximizing Anticancer Response with MPS1 and CENPE Inhibition Alongside Apoptosis Induction

doi: 10.3390/pharmaceutics16010056

Figure Lengend Snippet: CENPE and MPS1 are overexpressed in NSCLC lung cancer cell lines. mRNA expression of CENPE ( a ) and MPS1 ( c ) was determined by qRT-PCR in A549 and NCI-H460 cancer cell lines, and was compared to that in non-tumor HPAEpiC cells. Protein levels of CENPE ( b ) and MPS1 ( d ) were quantified by Western blotting assay, using α-tubulin as control. Data represent the mean ± SD of three independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The non-cancer cell line HPAEpiC (Human Pulmonary Alveolar Epithelial Cells; ScienCell Research Laboratories, San Diego, CA, USA) was grown in the same conditions as A549.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Micro-Immunotherapy Medicine 2LPAPI ® Displays Immune-Modulatory Effects in a Model of Human Papillomavirus Type-16 L1-Protein Capsid-Treated Human Peripheral Blood Mononuclear Cells and Antiproliferative Effects in a Model of Cervical Cancer Cells

doi: 10.3390/cancers16071421

Figure Lengend Snippet: MIM-1 and MIM-5 lowered the cellular viability/proliferative potential in a model of starved HPV-positive cervical cancer cells. Cell viability/proliferative potential of HeLa cells was assessed via AlamarBlue reagent under the experimental conditions presented in (see ) when treated with the vehicle (Veh.), MIM-1 or MIM-5. The results are presented as the mean percentages ± standard deviation (S.D.) of n = 6 replicates per condition, with the Veh. being set at 100%.

Article Snippet: HeLa cells (human cervix epithelioid carcinoma) were supplied by the European Collection of Authenticated Cell Cultures (ECACC) (ref: #93021013, ECACC, Salisbury, UK) and cultured according to the supplier’s recommendations.

Techniques: Standard Deviation

Journal: Pharmaceuticals

Article Title: Synthesis, Characterization and Antiproliferative Evaluation of Pt(II) and Pd(II) Complexes with a Thiazine-Pyridine Derivative Ligand †

doi: 10.3390/ph14050395

Figure Lengend Snippet: Dose–response curves of chemotherapeutics on cell viability of tumor and non-tumor cell lines. ( A ) U-937, ( B ) HL-60, ( C ) HeLa, ( D ) SK-OV-3 and ( E ) MCF10A cells were treated for 24 h with increasing concentrations (5, 10, 25, 50, 100 and 200 μM, as indicated) of the ligand PyTz, the Pt(II) (PtPyTz) and Pd(II) (PdPyTz) complexes, or the vehicle (DMF, control). Data represent means ± S.D. of six independent experiments and are expressed as a percentage of control values. * p < 0.05 compared to their corresponding control values. # p < 0.05 compared to their corresponding PtPyTz values.

Article Snippet: Human epithelial cervix carcinoma HeLa cell line (No. 93021013), human ovary adenocarcinoma SK-OV-3 cell line (No. 91091004), human histiocytic lymphoma U-937 cell line (No. 85011440), and human promyelocytic leukemia HL-60 15-12 cell line (No. 88120805) were purchased from the European Collection of Authenticated Cell Cultures (ECACC) (Dorset, U.K.).

Techniques: Control

Journal: Pharmaceuticals

Article Title: Synthesis, Characterization and Antiproliferative Evaluation of Pt(II) and Pd(II) Complexes with a Thiazine-Pyridine Derivative Ligand †

doi: 10.3390/ph14050395

Figure Lengend Snippet: Cytotoxicity (IC50 ± SD, µM) of the free ligand PyTz and their Pt(II) and Pd(II) complexes towards selected tumor and healthy (non-tumor) cell lines.

Article Snippet: Human epithelial cervix carcinoma HeLa cell line (No. 93021013), human ovary adenocarcinoma SK-OV-3 cell line (No. 91091004), human histiocytic lymphoma U-937 cell line (No. 85011440), and human promyelocytic leukemia HL-60 15-12 cell line (No. 88120805) were purchased from the European Collection of Authenticated Cell Cultures (ECACC) (Dorset, U.K.).

Techniques:

Journal: PLoS ONE

Article Title: Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

doi: 10.1371/journal.pone.0007174

Figure Lengend Snippet: (A) Cell-surface expression of CEACAM5 (CEA) and NIP-modified molecules on HeLa, HeLa CEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLa NIP ). (B) FACS analysis of CD69 expression by Jurkat EGFP , Jurkatα CEA-CIR-EGFP and Jurkatα NIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLa CEA or HeLa NIP ) for 16 hours.

Article Snippet: 293T cells (human embryo kidney epithelia; CRL-11268), HeLa cells (human cervix carcinoma; CCL-2), HT-1080 cells (human fibrosarcoma; CCL-121), MKN45 cells (human stomach adenocarcinoma; JCRB-0254), and MDA-MB231 cells (human breast adenocarcinoma; HTB-26) were grown in DMEM supplemented with 10% (vol/vol) heat-inactivated FCS, (Invitrogen Life Technologies, Gaithersburg, MD, USA), referred as to DMEM complete medium (DCM).

Techniques: Expressing, Modification, Labeling

Journal: PLoS ONE

Article Title: Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

doi: 10.1371/journal.pone.0007174

Figure Lengend Snippet: (A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, Jurkatα CEA-CIR-EGFP and Jurkatα NIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.

Article Snippet: 293T cells (human embryo kidney epithelia; CRL-11268), HeLa cells (human cervix carcinoma; CCL-2), HT-1080 cells (human fibrosarcoma; CCL-121), MKN45 cells (human stomach adenocarcinoma; JCRB-0254), and MDA-MB231 cells (human breast adenocarcinoma; HTB-26) were grown in DMEM supplemented with 10% (vol/vol) heat-inactivated FCS, (Invitrogen Life Technologies, Gaithersburg, MD, USA), referred as to DMEM complete medium (DCM).

Techniques: Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.

doi: 10.4161/cc.4.10.2046

Figure Lengend Snippet: Figure 2. A large portion of the N-terminal regulatory domain contributes to the degradation of cyclin A. (A) Promoter turn-off assays reveal different instability of N∆mutants. HtTA1 cells were transfected with plasmids expressing the indicated FLAG-tagged constructs. The cells were treated with buffer, Dox, or LLnL as indicated for 10 hr. Cell extracts were prepared and were subjected to immunoblotting with a monoclonal antibody against FLAG. (B) D-box deletion mutants are only partially stabilized. Cells were transfected with plasmids expressing the indicated FLAG-tagged constructs. Dox was added to the medium and the cells were harvested at the indicated time points. Cell extracts were prepared and the stability of FLAG-tagged proteins was examined by immunoblotting. (C) G2/M cell cycle delay induced by truncated cyclin A. Cells were cotransfected with plasmids expressing the indicated cyclin A constructs and a GFP-histone H2B marker. The cell cycle distribution of the transfected (GFP-positive) and untransfected (GFP-negative) cells was examined by flow cytometry. (D) Cell cycle delay induced by truncated cyclin A does not affect cyclin A degradation. FLAG-tagged cyclin A was expressed either alone or together with N∆157. The stability of cyclin A was analyzed as in (B).

Article Snippet: HtTA1 cells are HeLa cells (human cervical carcinoma) expressing the tTA tetracycline repressor chimera.34 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37 ̊C in 5% CO2.

Techniques: Transfection, Expressing, Construct, Western Blot, Marker, Flow Cytometry

Journal: Cell cycle (Georgetown, Tex.)

Article Title: The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.

doi: 10.4161/cc.4.10.2046

Figure Lengend Snippet: Figure 3. Cyclin A is ubiquitinated after extensive deletion of the N-terminal region. (A) In vivo ubiquitination of cyclin A. HtTA1 cells were cotransfected with plasmids expressing HA-Ub and the indicated FLAG-tagged constructs. Cells were treated with LLnL to inhibit the proteasome 6 hr before harvested. Cell extracts were prepared and 200 µg was subjected to immunoprecipitation with either control normal rabbit serum (NRS) or FLAG antiserum. The immunoprecipitates were then immunoblotted with antibodies against HA to detect ubiquitinated proteins. Expression of FLAG-tagged proteins was confirmed by immunoblotting with a monoclonal antibody against FLAG. Total cell lysates (10 µg) were loaded to indicate the inputs. (B) Relative ubiquitination of cyclin A mutants. Cells were cotransfected with plasmids expressing HA-Ub and various FLAG-cyclin A constructs as indicated. FLAG-cyclin B and a fusion protein containing cyclin A’s regulatory domain and cyclin B’s cyclin domain are expressed in lanes 11 and 10 respectively. Ubiquitination assays were preformed as in panel (A). Total cell lysates (top panel) and the FLAG-immunoprecipitates were immunoblotted for HA or FLAG as indicated.

Article Snippet: HtTA1 cells are HeLa cells (human cervical carcinoma) expressing the tTA tetracycline repressor chimera.34 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37 ̊C in 5% CO2.

Techniques: In Vivo, Ubiquitin Proteomics, Expressing, Construct, Immunoprecipitation, Control, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.

doi: 10.4161/cc.4.10.2046

Figure Lengend Snippet: Figure 4. Destruction of cyclin A in mitotic extracts is partially dependent on the D-box. (A) In vitro degradation of cyclin A in mitotic extracts. HtTA1 cells transfected with blank vectors (top two panels) or with plasmids expressing EMI1 were treated with nocodazole for 16 hr. Cell extracts were prepared and mixed with [35S]-labeled cyclin A produced in rabbit reticulocyte lysates. LLnL was applied in the reaction in the second panel. Samples were collected at the indicated time points, applied onto SDS-PAGE, and detected with PhosphorImagery. (B) Sequences outside the D-box contribute to the degradation of cyclin A in mitotic extracts. Cyclin A, cyclin B, and the indicated fusion proteins between cyclin A and cyclin B were expressed in rabbit reticulocyte lysates in the presence of [35S]-methionine. The reticulocyte lysates were mixed with mitotic cell extracts (Materials and Methods). Samples were harvested at the indicated time points, applied onto SDS-PAGE, and detected with PhosphorImagery. (C) Deletion of the D-box partially decreases degradation in mitotic extracts. Cyclin A and the indicated N-terminally truncation mutants were expressed in rabbit reticulocyte lysates and mixed with mitotic extracts as in panel (B). The [35S]-labeled proteins remaining in the reactions were quantified with PhosphorImagery (right panel). This experiment was repeated twice and similar relative rates of degradation between different mutants were obtained. The absolute rate of degradation varied slightly between different batches of mitotic extracts. Experiments that compared degradation rates in this study were performed together using the same batch of cell extracts.

Article Snippet: HtTA1 cells are HeLa cells (human cervical carcinoma) expressing the tTA tetracycline repressor chimera.34 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37 ̊C in 5% CO2.

Techniques: In Vitro, Transfection, Expressing, Labeling, Produced, SDS Page

Journal: Cell cycle (Georgetown, Tex.)

Article Title: The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.

doi: 10.4161/cc.4.10.2046

Figure Lengend Snippet: Figure 6. Characterization of a putative second D-box in cyclin A. (A) Alignment of the D-box sequences from human (Hs) cyclin B1 and cyclin A (DB1), and the putative second D-box (DB2) from human, cow (Bt), mouse (Mm), hamster (Ma) and rat (Rn). The RxxL motif is highlighted. (B) Schematic diagram of the cyclin A D-box mutants used in this study. The positions of DB1 and DB2 are shown to scale and the sequences of the RxxL motifs are indicated. (C) The relative stability of D-box mutants in vivo. HtTA1 cells were transfected with plasmids expressing various D-box mutants as indicated. Promoter turn-off experiments were performed as described in Figure 2B. The proteasome inhibitor LLnL was included in lane 5. (D) Ubiquitination of D-box mutants. In vivo ubiquitination assays were performed with cyclin A the indicated D-box mutants as described in Fig. 3A. (E) The putative second D-box only contributes weakly to cyclin A destruction. Cyclin A and the indicated D-box mutants were expressed in rabbit reticulocyte lysates and were sub- jected to in vitro destruction assays as described in Figure 4B.

Article Snippet: HtTA1 cells are HeLa cells (human cervical carcinoma) expressing the tTA tetracycline repressor chimera.34 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37 ̊C in 5% CO2.

Techniques: In Vivo, Transfection, Expressing, Ubiquitin Proteomics, In Vitro

Journal: Cell cycle (Georgetown, Tex.)

Article Title: The N-terminal regulatory domain of cyclin A contains redundant ubiquitination targeting sequences and acceptor sites.

doi: 10.4161/cc.4.10.2046

Figure Lengend Snippet: Figure 9. The three lysine residues in the D-box region are involved in controlling cyclin A degradation. (A) Schematic diagram of the KR mutants used in this study. Wild type cyclin A is shown on the top and the positions of the three N-terminal lysine residues are shown (only sequences up to the cyclin box are shown). (B) Cyclin A was degraded slower when all three N-terminal lysine residues were substituted. The degradation of cyclin A and the indicated mutants was examined by addition of Dox as described in Figure 2B. (C) The KR mutants are still ubiquitinated. Cyclin A and the indicated mutants were expressed in HtTA1 cells and subjected to ubiq- uitination assay as described in Figure 3A.

Article Snippet: HtTA1 cells are HeLa cells (human cervical carcinoma) expressing the tTA tetracycline repressor chimera.34 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37 ̊C in 5% CO2.

Techniques: