Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Association between clinicopathological factors and the expression of β-catenin.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Expression of β-catenin in BC tissues and cell lines. The expression of β-catenin was determined in 32 paired BC tissues at the (A) mRNA and (B) protein levels were determined by RT-qPCR and western blot analysis, respectively. (C) The expression of β-catenin was analyzed in BC tissues by immunohistochemistry. Magnification, ×200. (D) Score analyses of the immunohistochemistry results (n=32 vs. 32). The expression levels of β-catenin in the BC MCF-10A, MDA-MB-468 and T47D cell lines and MCF-7 cells at the (E) mRNA and (F and G) protein levels were determined by RT-qPCR and western blot analysis, respectively. All data are presented as the mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. adjacent tissues or normal cells MCF-10A. BC, breast cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Viability of BC cell lines and the expression of β-catenin are regulated by cisplatin and siRNA interference. The viability of (A) T47D and (B) MCF-7 cells was inhibited by cisplatin at different concentration (20, 40, 80 and 160 nM) determined by CCK-8 assays for 24 h. (C) MCF-7 cells were either not transfected or transfected with control siRNA or siR-β-catenin. At 24 h post transfection, cells were lysed and β-catenin expression was determined by western blot analysis. The viability of (D) T47D and (E) MCF-7 cells was suppressed by the combination of cisplatin (80 nM) and siR-β-catenin for 24 h. Each sample was analyzed in triplicate and was normalized to the control. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer; siRNA, small interfering RNA.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Expressing, Concentration Assay, CCK-8 Assay, Transfection, Western Blot, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Migration and invasion abilities of BC cells are suppressed by combined treatment with cisplatin and siR-β-catenin. The migratory abilities of (A and B) T47D cells and (C and D) MCF-7 cells treated with cisplatin and siR-β-catenin was evaluated using Transwell assays. The invasive ability of (E and F) T47D cells and (G and H) MCF-7 cells treated with cisplatin and siR-β-catenin was determined using Transwell assays. All data are presented as the mean ± standard error of the mean of 3 independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer; siRNA, small interfering RNA.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Migration, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Expression levels of CD44/54 in BC cells are analyzed by flow cytometry. (A) T47D cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin were analyzed by FACS and the fluorescence intensities of CD44/CD54 were obtained. (B) Statistical analysis of the expression of CD44/CD54 in T47D cells. (C) The expression of CD44/CD54 in MCF-7 cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin were analyzed by FACS. (D) Statistical analysis of the expression of CD44/CD54 in MCF-7 cells. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer; siRNA; small interfering RNA; CD44, CD44 antigen; CD54, intercellular adhesion molecule 1.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Cell cycle distribution of BC cells treated with the combination of cisplatin and siR-β-catenin detected by flow cytometry. (A) The cell cycle distribution of T47D cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin was determined by flow cytometry. (B) Statistical analysis of the cell cycle analysis results of T47D cells. (C) The cell cycle distribution of MCF-7 cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin was determined by flow cytometry. (D) Statistical analysis of the cell cycle analysis results of T47D cells. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Flow Cytometry, Cell Cycle Assay

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Cisplatin- and siR-β-catenin-induced apoptosis of BC cells is measured using flow cytometry. (A) T47D cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin were examined by flow cytometry. (B) Statistical analysis of apoptosis assay results in T47D cells. (C) The levels of apoptosis in MCF-7 cells treated with cisplatin, siR-β-catenin and the combination of cisplatin and siR-β-catenin was determined by flow cytometry. (D) Statistical analysis of apoptosis assay results in MCF-7 cells. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer; siRNA, small interfering RNA.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Flow Cytometry, Apoptosis Assay, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Levels of apoptosis in BC cells induced by treatment with cisplatin and siR-β-catenin in combination are analyzed using Hoechst 33258 staining. (A and B) Apoptosis was significantly increased in T47D cells treated with the combination of cisplatin and siR-β-catenin. (C and D) Apoptosis was significantly increased in MCF-7 cells treated with the combination of cisplatin and siR-β-catenin. Nuclear morphological changes were observed under a fluorescence microscope. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001, vs. control. BC, breast cancer; siRNA, small interfering RNA.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Staining, Fluorescence, Microscopy, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β-catenin silencing

doi: 10.3892/ijmm.2020.4543

Figure Lengend Snippet: Proteins of the β-catenin signaling pathway and apoptosis-associated proteins are regulated by treatment with cisplatin and siR-β-catenin in combination. (A) The expression levels of signaling pathway proteins β-catenin, c-Myc and cyclin D1 were suppressed by the combination of cisplatin and siR-β-catenin in MCF-7 cells. (B) Statistical analysis of the expression levels of β-catenin, c-Myc and cyclin D1 in MCF-7 cells. (C) The levels of apoptosis-associated proteins caspase-3 and caspase-9 were increased by the treatment of combination of cisplatin and siR-β-catenin in MCF-7 cells. (D) Statistical analysis of caspase-3 and caspase-9 expression in MCF-7 cells. All data are presented as mean ± standard error of the mean. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. BC, breast cancer; siRNA, small interfering RNA; c-Myc, MYC proto-oncogene, BHLH transcription factor.

Article Snippet: Once the cells reached 95% confluence, they were trans-fected with a SignalSilence ® β-catenin siRNA II (siR-β-catenin; cat. no. 6238; Cell Signaling Technology, Inc.) or unconjugated SignalSilence ® control siRNA (cat no. 6568; Cell Signaling Technology, Inc.) with Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Expressing, Small Interfering RNA

Journal: Scientific Reports

Article Title: Polyphyllin I suppresses human osteosarcoma growth by inactivation of Wnt/β-catenin pathway in vitro and in vivo

doi: 10.1038/s41598-017-07194-9

Figure Lengend Snippet: PPI suppressed osteosarcoma cells by specifically inactivating Wnt/β-catenin signaling pathway. ( A ) RT-PCR analysis of β-catenin expression level in matched human osteosarcoma tissues (tumors) and adjacent noncancerous tissues (normal) from 3 patients. ( B ) 143-B cells and ( C ) HOS cells were respectively treated with 0.8 μM PPI for indicated times, and expressions of test proteins were examined by western blotting analysis, β-actin was used as loading control, and the full-length blots were included in the supplementary information file as Figures and . 143-B cells were pretreated with 4 μM CHIR9902 (the specific GSK-3β inhibitor) for 24 h before exposed to 0.8 μM PPI for another 48 h, the combined CHIR and PPI treatments result in rescued ( D ) active β-catenin expression (the full-length blots were included in the supplementary information file as Figure ) and cell viability ( E ) compared to the PPI treatment alone in 143-B osteosarcoma cells. 143-B cells were transfected with either small interfering RNA-targeting β-catenin (si-β-catenin) or si-control for 48 h before exposed to 0.8 μM PPI for another 24 h, si-β-catenin potentiated PPI induced ( F ) down-regulation of active β-catenin expression (the full-length blots were included in the supplementary information file as Figure ), ( G ) decrease of cell viability and ( H ) inhibition of migration of 143-B osteosarcoma cells induced by PPI. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the control.

Article Snippet: Briefly, 143-B cells were co-transfected with either small interfering RNA-targeting β-catenin (100 nM si-β-catenin) or 100 nM si-control for 72 h, using Lipofectamine 2000 (Invitrogen). β-catenin siRNA II (#6238) was bought from Cell Signaling Technology (Danvers, USA). si-control was designed and produced by Shanghai GenePharma Co.,Ltd (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Small Interfering RNA, Inhibition, Migration

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Generation of BHV-1 ORF2 mutants. (A) Amino acid sequence of ORF2. NLS (underlined), 15 putative phosphorylation sites (gray shaded amino acids), and 5 consensus protein kinase A (PKA) and/or PKC phosphorylation sites (gray shaded amino acids that have white lettering) are shown. The plus signs denote every 10th amino acid in ORF2. (B) ORF2 coding sequences (BamHI-SalI) were cloned into the pUC57 vector, and transposon linker insertion reactions were performed as described in Materials and Methods. Initial mapping was performed by restriction digestion, and the precise location of the transposon insertion was confirmed by sequencing. Vertical lines with numbers indicate nucleotide positions of transposon insertions. The relative position of the consensus nuclear localization signal (NLS) is denoted by the white rectangle. (C) The transposon mutants and the two phosphorylation mutants were cloned into the pCMV-Tag-2B vector and transfected into Neuro-2A cells. At 48 h after transfection, cells were collected and lysed using hypotonic buffer as described in Materials and Methods. After centrifugation, the supernatant was removed. The nuclei and other cellular debris were suspended in RIPA buffer, and the solubilized proteins were collected after centrifuging the residual debris; this solubilized fraction was designated the pellet. Detection of Flag-tagged ORF2 mutants was performed using an Anti-Flag antibody. A β-actin antibody was used to confirm that equal amounts of protein were loaded in each lane. A histone 3 antibody was used to identify nuclear proteins in the supernatant or pellet fraction. ORF2 is predicted to migrate as a 19-kDa protein, and the black circle denotes the position of this protein. The arrow denotes the higher-molecular-weight ORF2-specific bands that migrated at approximately 30 kDa, which were detected in certain samples. The transposon mutants are predicted to migrate as a 22-kDa protein and are denoted by a closed triangle. The location of a nonspecific band in D is denoted by the asterisk. The bracket denotes the position of the ORF2-specific bands that migrated slower than expected. For each lane, 100 μg protein was loaded. (D) Neuro-2A cells were transfected with the designated plasmids and the respective samples collected at 48 h after transfection as described above. These results are representative of more than 3 independent experiments.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Transfection, Centrifugation, Molecular Weight

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Localization of ORF2 mutants in Neuro-2A cells. Neuro-2A cells were transfected with 4 μg of the designated plasmids that express a Flag-tagged ORF2 or ORF2 mutants. Cultures were prepared for confocal microscopy at 48 h after transfection as described in Materials and Methods. ORF2+ cells were identified using the anti-Flag antibody (red), or DAPI was used to visualize the nucleus (blue). Differential interference contrast (DIC) was used to show the unstained cells. The images are representative of more than 3 experiments.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Transfection, Confocal Microscopy

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Inhibition of cold shock-induced DNA laddering by ORF2 mutants. (A) Neuro-2A cells were transfected with 4 μg of the plasmid expressing N-terminally Flag-tagged ORF2 or the designated ORF2 transposon mutants. Cells were cold shocked at 4°C for 1 h and allowed to recover at 37°C for 3 h. Neuro-2A cells transfected with the empty vector were used as a negative control, while Bcl-2- or ORF2-expressing cells were used as positive controls. The agarose gel images are representative of 5 independent experiments. (B) The relative amounts of fragmented DNA in each lane in A were quantified using a Bio-Rad Molecular Imager FX. The average of 5 independent experiments is shown with standard deviation. (C) Neuro-2A cells transfected with the empty vector, ORF2, ORF2-ΔNLS, ORF2-P, or ORF2-AP were cold shocked as in A, and fragmented DNA was quantified as described in B. An asterisk denotes significant differences (P < 0.05) from the wt samples (panel B) or (empty vector) (panel C) as determined by the Student t test.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Inhibition, DNA Laddering, Transfection, Plasmid Preparation, Expressing, Negative Control, Agarose Gel Electrophoresis, Standard Deviation

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Survival of Neuro-2A cells after transfection with ORF2 mutants. Neuro-2A cells were cotransfected with 2 μg of the pCMV-β-Gal plasmid and 2 μg of the designated ORF2 expression plasmids. Cells were cold shocked for 2 h, and the β-Gal assay was performed as described in Materials and Methods. The number of β-Gal+ cells in cultures expressing the blank vector was set to 100%. The number of blue cells in cultures transfected with the blank vector was divided by the number of blue cells in cultures transfected with ORF2 or ORF2 mutants to calculate the fold difference. The results are an average of three independent experiments. An asterisk denotes significant differences (P < 0.05) from the wt ORF2 results as determined by the Student t test.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Induction of caspase 3 cleavage by ORF2 transposon mutants. Neuro-2A cells were transfected for 24 h and incubated in EMEM with 2% FCS for 12 h, and then cold shock-induced apoptosis was performed as described in Materials and Methods. Cells were then processed for immunofluorescence as described in Materials and Methods. Mouse anti-Flag antibody (red) was used to detect Flag-tagged ORF2 and the designated transposon mutants. Rabbit anti-cleaved caspase 3 antibody (green) was used to detect activated cleaved caspase 3. DAPI (blue) was used to stain the nucleus.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Transfection, Incubation, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Identification of ORF2 sequences that interfere with Notch1-mediated trans-activation of the bICP0 early promoter. Neuro-2A cells were cotransfected with the bICP0 E promoter construct (EP-172), a CMV promoter plasmid expressing the Notch1 intracellular domain, and the designated ORF2-expressing constructs. At 48 h posttransfection, cells were collected and processed for CAT activity as described in Materials and Methods. The CAT activity of cells transfected with the control CAT vector was set to 1. All other values are expressed as fold activation with respect to the control. These studies are the average of at least three independent experiments. An asterisk denotes significant differences (P < 0.05) from the wt ORF2 samples as determined by the Student t test.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Activation Assay, Construct, Plasmid Preparation, Expressing, Activity Assay, Transfection

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Identification of ORF2 sequences that interfere with Notch1-mediated trans-activation of productive infection. Neuro-2A cells were cotransfected with the gCblue virus, a CMV promoter plasmid expressing the intracellular domain of Notch1 and the designated ORF2 constructs. At 48 h after transfection, cells were fixed and a β-Gal assay was performed as described in Materials and Methods. The number of β-Gal+ cells in cultures expressing the empty vector was set to 1. The number of blue cells in cultures transfected with the empty vector was divided by the number of blue cells in cultures transfected with Notch1 or cotransfected with Notch1 and ORF2 or ORF2 mutants to calculate the fold difference. The results are an average of at least three independent experiments. An asterisk denotes significant differences (P < 0.05) from the wt ORF2 samples (empty vector) as determined by the Student t test.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Activation Assay, Infection, Plasmid Preparation, Expressing, Construct, Transfection

Journal: Journal of Virology

Article Title: Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter ^▿

doi: 10.1128/JVI.05478-11

Figure Lengend Snippet: Induction of caspase 3 cleavage by ORF2 transposon mutants. Neuro-2A cells were transfected for 24 h and incubated in EMEM with 2% FCS for 12 h, and then cold shock-induced apoptosis was performed as described in Materials and Methods. Cells were then processed for immunofluorescence as described in Materials and Methods. Mouse anti-Flag antibody (red) was used to detect Flag-tagged ORF2 and the designated transposon mutants. Rabbit anti-cleaved caspase 3 antibody (green) was used to detect activated cleaved caspase 3. DAPI (blue) was used to stain the nucleus.

Article Snippet: Cells were permeabilized with cold 100% ethanol at −20°C for 5 min. After washing three times with 1× phosphate-buffered saline (PBS), slides were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h and then incubated with the mouse anti-Flag antibody (Sigma F1804) (1:250) (Flag-ORF2) or rabbit anti-cleaved caspase 3 (Cell Signaling 6238S) (1:250) for 2 h at room temperature.

Techniques: Transfection, Incubation, Immunofluorescence, Staining