Journal: Virology

Article Title: Human Pegivirus (HPgV; formerly known as GBV-C) inhibits IL-12 dependent natural killer cell function

doi: 10.1016/j.virol.2015.07.008

Figure Lengend Snippet: Extracellular vesicles (EVs) containing recombinant HPgV E2 protein inhibit IL-12-mediated signaling and deliver E2 to NK cells. IFNγ release by the NK cell line (NK92MI) following incubation with CHO cell culture supernatant EVs containing recombinant HPgV E2-human Fc fusion protein or human Fc protein (A). Cells were incubated 18 h prior to stimulation with IL-12 (p value for T test shown). E2-Fc and Fc concentration used was 50 µg/ml. EVs from cell culture supernatant fluids of cells expressing HPgV E2-Fc contained E2, while EVs from CHO cells expressing just Fc protein did not (B). NK92MI cells were incubated overnight with HPgV E2 positive EVs and the cells examined for surface or total cellular E2 protein following permeabilization using flow cytometry (C). The mean fluorescence intensity (MFI) of surface and total HPgV E2 in the cells is summarized in the graph (C).

Article Snippet: Recombinant E2 protein (40 μg) was incubated with or without active human Tyk2 (250 ng) using an in vitro kinase assay as recommended by the manufacturer (Signal Chem).

Techniques: Recombinant, Incubation, Cell Culture, Concentration Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Translational Oncology

Article Title: Specific Pomegranate Juice Components as Potential Inhibitors of Prostate Cancer Metastasis 1 2

doi:

Figure Lengend Snippet: Effect of L + E + P on genes involved in cell growth, cell adhesion, and migration of hormone-independent prostate cancer cells. (A) Schematic summary of the effects of L + E + P on gene and miRNA expression in hormone-independent prostate cancer cells. (B) The mRNA levels of CCNE2, ANLN, CCNB1, DTL, HMMR, TWIST, ROCK2, CDC25B, BCL2, NEXN, CDK6, COL1A1, FSCN1, EZH2, PTEN, CDKN1A, and CLDN1 were determined by using qPCR with RNA extracted from PC3 cells treated with L + E + P at 8 µg/ml for 12 hours.

Article Snippet: PC3 cells (80–90% confluent) were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocols; 40 nM E-cadherin chimera small-interfering RNA (siRNA) (Abnova, Taiwan, China) was transfected, and 2 µg/ml pcDNA3.1 HMMR vector, pcDNA4.1 TWIST vector, or pcDNA3 CCNE2 vector (Addgene plasmid 19935; Addgene, Cambridge, MA) was transfected.

Techniques: Migration, Expressing

Journal: Translational Oncology

Article Title: Specific Pomegranate Juice Components as Potential Inhibitors of Prostate Cancer Metastasis 1 2

doi:

Figure Lengend Snippet: Gene Analysis of the Effects of L + E + P on PC3 Cells.

Article Snippet: PC3 cells (80–90% confluent) were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocols; 40 nM E-cadherin chimera small-interfering RNA (siRNA) (Abnova, Taiwan, China) was transfected, and 2 µg/ml pcDNA3.1 HMMR vector, pcDNA4.1 TWIST vector, or pcDNA3 CCNE2 vector (Addgene plasmid 19935; Addgene, Cambridge, MA) was transfected.

Techniques: Migration, Binding Assay

Journal: Translational Oncology

Article Title: Specific Pomegranate Juice Components as Potential Inhibitors of Prostate Cancer Metastasis 1 2

doi:

Figure Lengend Snippet: Effect of L + E + P on Cancer-Related miRNAs.

Article Snippet: PC3 cells (80–90% confluent) were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocols; 40 nM E-cadherin chimera small-interfering RNA (siRNA) (Abnova, Taiwan, China) was transfected, and 2 µg/ml pcDNA3.1 HMMR vector, pcDNA4.1 TWIST vector, or pcDNA3 CCNE2 vector (Addgene plasmid 19935; Addgene, Cambridge, MA) was transfected.

Techniques:

Journal: Translational Oncology

Article Title: Specific Pomegranate Juice Components as Potential Inhibitors of Prostate Cancer Metastasis 1 2

doi:

Figure Lengend Snippet: Mechanistic study of the effect of L + E + P on cell growth, cell adhesion, and migration of hormone-independent prostate cancer cells. (A) PC3 cells were transfected with pcDNA3 CCNE2 vector (2 µg/ml) and treated with L + E + P at 8 µg/ml 24 hours after transfection. Cell number was counted at a 48-hour time point. (B) Immunoblot analysis for E-cadherin with protein extracts from PC3 cells treated with L + E + P at 8 µg/ml for 24 hours. (C) PC3 cells were transfected with 40 nM E-cadherin siRNA. Twenty-four hours after transfection, cells were treated with L + E + P at 8 µg/ml for 12 hours and adhesion assay was performed. PC3 cells were transfected with 2 µg/ml (D) pcDNA3.1 HMMR vector or (E) pcDNA4.1 TWIST vector and treated with L + E + P at 8 µg/ml 24 hours after transfection. Migrated distance was determined at a 48-hour time point. Bars represent SEM. **P < .01; *P < .05.

Article Snippet: PC3 cells (80–90% confluent) were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocols; 40 nM E-cadherin chimera small-interfering RNA (siRNA) (Abnova, Taiwan, China) was transfected, and 2 µg/ml pcDNA3.1 HMMR vector, pcDNA4.1 TWIST vector, or pcDNA3 CCNE2 vector (Addgene plasmid 19935; Addgene, Cambridge, MA) was transfected.

Techniques: Migration, Transfection, Plasmid Preparation, Western Blot, Cell Adhesion Assay

Journal: Ecotoxicology and environmental safety

Article Title: The influence of phenolic environmental estrogen on the transcriptome of uterine leiomyoma cells: A whole transcriptome profiling-based analysis.

doi: 10.1016/j.ecoenv.2021.111945

Figure Lengend Snippet: Fig. 4. The effects of 10.0 μmol/L BPA, 32.0 μmol/L NP on genes and proteins expression in uterine leiomyoma cells. (a) Hierarchically clustered heatmap of differentially expressed genes associated with cell cycle after BPA and NP exposure. (b) E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression by q-PCR presented as relative changes. There were increased E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 genes expression in uterine leiomyoma cells treated with10.0 μmol/L BPA, 32.0 μmol/L NP for 48 h compared to control group (*P < 0.05). (c) Representative western blots and (d) band intensity bar graphs of proteins. There were significantly (*P < 0.05) higher expression levels of E2F1, CCND1, CCNE2, MCM2, MCM3, MCM4, MCM5, MCM6 in the cells treated with BPA and NP for 48 h compared to controls. The western band images shown are representative of three independent experiments and the data were expressed as mean ± SE done in three independent experiments.

Article Snippet: The following primary antibodies were used for the western blotting: E2F1 Monoclonal Antibody (66515-1-Ig; Proteintech; diluted 1:1000); Cyclin D1 Monoclonal Antibody (60186-1-Ig; Proteintech; diluted 1:5000); Cyclin E2 Polyclonal Antibody (11935-1-AP; Proteintech; diluted 1:500); MCM2 Polyclonal Antibody (10513-1-AP; Proteintech; diluted 1:1500); MCM3 Polyclonal Antibody (15597-1-AP; Proteintech; diluted 1:1000); MCM4 Polyclonal Antibody (13043-1-AP; Proteintech; diluted 1:600); MCM5 Polyclonal Antibody (11703-1-AP; Proteintech; diluted 1:1000); MCM6 Polyclonal Antibody (13347-2-AP; Proteintech; diluted 1:8000); phospho-Rb monoclonal (ab184796; abcam; diluted 1:1000); phospho-PI3K polyclonal (ab182651; abcam; diluted 1:500); phospho- AKT polyclonal (ab38449; abcam; diluted 1:500).

Techniques: Expressing, Control, Western Blot

Journal: Cancer cell

Article Title: A critical role for notch signaling in the formation of cholangiocellular carcinomas.

doi: 10.1016/j.ccr.2013.04.019

Figure Lengend Snippet: Figure 2. Notch Signaling Leads to Dysregulated Expression of Cyclin E and Genetic Instability (A) b-catenin staining (for the detection of cell size) was performed on liver sections from wild-type and NotchIC::AlbCre mice at 12 weeks of age. (B) Quantification of cell size in the indicated mouse strains was done after b-catenin staining to visualize the cell surface at 12 weeks of age. Five hundred cells on average were analyzed using a photometric system. (C) Three hundred visual fields were counted on average to determine the number of hepatocytes in the livers of the indicated mouse strains at the age of 12 weeks. (D) The DNA content of hepatocytes of the indicated mouse strains was determined by microphotometry analysis of Feulgen-stained liver sections at the age of 11 weeks. (E and F) Quantification of the number of BrdU and phospho-Histone-H3-positive hepatocytes after induction of liver regeneration in wild-type and NotchIC::AlbCre double transgenic mice at the age of 10 weeks. For BrdU staining, an average of 900–2000 cells, and for HH3 staining at least 700 cells from up to (legend continued on next page)

Article Snippet: Transfections were performed using siRNA at a concentration of 20 nM for Notch 1 si RNA; sc-36095 and Notch 3 siRNA; sc-37135 from Santa Cruz. siRNA in a concentration of 40 nM was used for cyclin E1 and cyclin E2 knockdown.

Techniques: Expressing, Staining, Transgenic Assay, BrdU Staining

Journal: Cancer cell

Article Title: A critical role for notch signaling in the formation of cholangiocellular carcinomas.

doi: 10.1016/j.ccr.2013.04.019

Figure Lengend Snippet: Figure 3. Expression of Notch ICD in Mouse Liver Leads to Formation of Progenitor Cell Derived Cholangiocellular Carcinomas (A) H&E stained liver sections of NICD:AlbCre mice. Immunohistochemical staining of NotchIC::AlbCre livers at the age of nine months using antibodies against CK7, CK19, CK17, CK8-18, CD34, and CD34/Cytokeratin double staining. (B) NotchIC::Alb Cre livers at the age of 9 months were minced and injected subcutaneously into nude mice. The growth of the resulting tumors was monitored. (C) H&E and immunohistochemical analysis of the explanted tumor tissue using CK7 and CK17 antibodies 3 weeks after implantation of tumor cells. (D) Growth curves of tumors after s.c. injection of progenitor cells of the indicated genotypes: progenitor cells expressing c-Myc and Akt, progenitor cells expressing the intracellular domain of notch and a control shRNA (ICN-shCtrl.), progenitor cells expressing ICN together with a shRNA targeting cyclin E (ICN- shCyc.E), progenitors that only express shRNA control (shCtrl.), and progenitor cells expressing only shRNAs that target cyclin E (shCyc.E-A/B). (E)H&E stainedtissuederivedfromsubcutaneouslygrowing tumorsthatoriginatedfromNICDexpressingbipotentialprogenitorseithertransducedwithICN(ICN)and a shRNA control (ICN-shCtrl) or with Myc/Akt. The circle indicates atypical fused glands with hyperchromatic and irregular nuclei growing in a desmoplastic stroma. See also Figure S2.

Article Snippet: Transfections were performed using siRNA at a concentration of 20 nM for Notch 1 si RNA; sc-36095 and Notch 3 siRNA; sc-37135 from Santa Cruz. siRNA in a concentration of 40 nM was used for cyclin E1 and cyclin E2 knockdown.

Techniques: Expressing, Derivative Assay, Staining, Immunohistochemical staining, Double Staining, Injection, Control, shRNA

Journal: Cancer cell

Article Title: A critical role for notch signaling in the formation of cholangiocellular carcinomas.

doi: 10.1016/j.ccr.2013.04.019

Figure Lengend Snippet: Figure 4. Notch Signaling Leads to the Transformation of Bipotential Hepatic Progenitors through Dysregulation of Cyclin E Expression (A) Immunohistochemical staining of wild-type and NotchIC::AlbCre livers at the age of 9 months using a cyclin E antibody. (B) Livers from wild-type controls or tumor tissue derived from 9-month-old NotchIC::AlbCre mice were stained with a gH2AX antibody to determine the number of nuclei that showed signs of DNA damage. The plot shows the staining results of at least 300 cells per mouse strain. An example of NotchIC::AlbCre and wild- type nuclei is shown in a 4003 magnification on the right side. (C) Representative examples of gH2AX and cyclin E stained transplanted mouse CCCs and wild-type liver control. (D) Statistical analysis of gH2AX foci in MzChA1 cells and after depletion of cyclin E expression through siRNA-mediated knockdown of cyclin E1, cyclin E2, or both. (E) MzChA1, TFK1, Hep3B, and HepG2 cells were transfected with the indicated plasmids and relative luciferase activity was measured after 48 hr. Scale bars represent mean values ± SEM. *p < 0.05; **p < 0.001.

Article Snippet: Transfections were performed using siRNA at a concentration of 20 nM for Notch 1 si RNA; sc-36095 and Notch 3 siRNA; sc-37135 from Santa Cruz. siRNA in a concentration of 40 nM was used for cyclin E1 and cyclin E2 knockdown.

Techniques: Transformation Assay, Expressing, Immunohistochemical staining, Staining, Derivative Assay, Control, Knockdown, Transfection, Luciferase, Activity Assay

Journal: Cancer cell

Article Title: A critical role for notch signaling in the formation of cholangiocellular carcinomas.

doi: 10.1016/j.ccr.2013.04.019

Figure Lengend Snippet: Figure 5. The Notch Signaling Pathway Is Activated in Human CCC (A) TFK1, MzChA1, EGI1 cells, a CCC primary culture (SZ1), and HeLa cells were lysed and the expression levels of Notch 1, cleaved Notch, Jagged 1 and HES1 were analyzed by western blotting. (B) Comparison of cleaved Notch and HES1 levels in human CCC cell lines and tumor tissue derived from NotchIC::AlbCre mice. Actin and GAPDH were used as loading controls. (C) Fifty-six different human CCCs were screened for Notch 1, 2, 3, or 4 expression using a tissue microarray. (D) Analysis of the staining intensities of the tissue microarray displayed is the percentage of tumors that express the Notch receptors 1–4 in the cyto- plasm as well as in the nucleus in the 56 CCC samples tested. (E) Immunohistochemical analysis of 56 human CCCs shows a strong expression of cyclin E1 compared to wild-type livers. See also Figure S3 and Table S1.

Article Snippet: Transfections were performed using siRNA at a concentration of 20 nM for Notch 1 si RNA; sc-36095 and Notch 3 siRNA; sc-37135 from Santa Cruz. siRNA in a concentration of 40 nM was used for cyclin E1 and cyclin E2 knockdown.

Techniques: Expressing, Western Blot, Comparison, Derivative Assay, Microarray, Staining, Immunohistochemical staining

Journal: Cancer cell

Article Title: A critical role for notch signaling in the formation of cholangiocellular carcinomas.

doi: 10.1016/j.ccr.2013.04.019

Figure Lengend Snippet: Figure 6. Notch Signaling as a Therapeutic Target in CCC (A) Western blot expression analysis of Notch 1, HES1, cyclin E, p21, p53, and p27 after siRNA- mediated knockdown of Notch 1 in MzChA1 cells. (B) Determination of cell death (sub G1 fraction) by FACS scan analysis in MzChA1 cells in which Notch1 expression was reduced through siRNA- mediated knockdown. (C) MzChA1 cells were treated with DAPT (10 mM) for the indicated periods of time. At different time points cells were lysed and the expression levels of Notch 1, cleaved Notch 1, and HES1 were analyzed by western blotting. (D) Caspase 3/7 activity in DAPT-treated MzChA1 cells at the indicated time points. The y-axis de- scribes the relative caspase activity in percentages as compared to the DMSO control treated cells. (E) Primary human CCC cells (SZ1) were trans- planted under the skin of nu/nu mice (ten mice for each group). When the tumors reached a size of approximately 150–200 mm3, mice were treated with DAPT at a concentration of 50 mg/kg every 72 hr or with DMSO, which was used to dissolve DAPT (control). (F) Western blot analysis of the expression levels of cyclin E1, p21, p27, p53, and actin in tumor tissue lysates from DAPT treated or untreated mice. Scale bars represent mean values ± SEM. See also Figure S4.

Article Snippet: Transfections were performed using siRNA at a concentration of 20 nM for Notch 1 si RNA; sc-36095 and Notch 3 siRNA; sc-37135 from Santa Cruz. siRNA in a concentration of 40 nM was used for cyclin E1 and cyclin E2 knockdown.

Techniques: Western Blot, Expressing, Knockdown, Activity Assay, Control, Concentration Assay

Journal: Thrombosis Journal

Article Title: Dimethyl fumarate inhibits antibody-induced platelet destruction in immune thrombocytopenia mouse

doi: 10.1186/s12959-021-00314-6

Figure Lengend Snippet: Cell cycle analysis after DMF treatment. RAW264.7 cells were treated with different concentrations of DMF (1, 10, 50,100 μM) or vehicle to detect cell cycle using EdU staining by flow cytometry ( A ) and the expression of cyclin D1 and E2 by western blot ( B ). Compared with 0, * P < 0.05; ** P < 0.01; *** P < 0.001 (mean ± SD, n = 3)

Article Snippet: Then, the membrane was incubated with antibodies against Bcl-2 (Cell Signaling Technology), Bax (Cell Signaling Technology), Cleaved Caspase3 (Cell Signaling Technology), cyclin D1 (Proteintech) and cyclin E2 (Affinity Biosciences) and subsequent with HRP-bound secondary antibody.

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Expressing, Western Blot