Journal: The American Journal of Pathology

Article Title: Fusion Tyrosine Kinase NPM-ALK Deregulates MSH2 and Suppresses DNA Mismatch Repair Function

doi: 10.1016/j.ajpath.2011.03.045

Figure Lengend Snippet: NPM-ALK suppresses MMR function in vitro. A: Tet-on HEK-293/NPM-ALK were grown in three specified doxycycline concentrations (0, 100 ng/mL, and 400 ng/mL) for 24 hours, and then 6TG was added for an additional 48 hours. The WST-1 assay was used to compare relative cell viability/proliferation, and the resulting colormetric response was measured with a μQuant BioTeck plate reader. Each experiment was plated in quadruplicate. Figure 2A shows a representative experiment. The results showed that doxycycline-induced expression of NPM-ALK in the Tet-on HEK293/NPM-ALK cells resulted in a significant increase in cell viability. Data are represented as mean ± SEM. Stars denote statistical difference between 0 ng/mL doxycycline and doxycycline-induced cells using one-way analysis of variance and the Newman-Keuls multiple comparison test (GraphPad Prism); *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. = not significant. Comparing the 100- and 400-ng/mL data, differences were statistically different (P < 0.05) at all doses, with the two highest reaching greater significance (3.125 μmol/L, P < 0.001; 1.56 μmol/L, P < 0.01). B: Tet-on HEK293/NPM-ALK cells were transiently transfected with the pCAR-OF vector, which carries cDNA encoding β-galactosidase placed out of frame by a (CA)29 repeat. Four hours after transfection, the medium was supplemented by either 400 ng/mL doxycycline (with NPM-ALK expression) or 0 ng/mL doxycycline (no NPM-ALK). Seventy-two hours after transfection, the cell lysates were assayed for β-galactosidase activity, an indication of suppressed MMR function. Cells with NPM-ALK expression showed a significantly higher level of β-galactosidase activity compared with cells with no expression of NPM-ALK. C: Tet-on HEK293/NPM-ALK cells were subjected to clonal expansion for 10 days in the presence (1000 ng/mL doxycycline at the initial concentration) or absence (0 ng/mL doxycycline) of NPM-ALK expression. DNA was harvested and MSI tested at both mono- and di-nucleotide microsatellites. MSI was detected at NR-27, which is also affected in ALK+ALCL cell lines. Shaded area represents the location of the predominant peaks in MMR-proficient cells.

Article Snippet: MMR Functional Assay Using 6TG The sensitivity of cells to 6-thioguanine (6TG) was tested in 96-well format, and the resulting cell viability was assayed using the WST-1 cell proliferation reagent (Clontech) with the absorbance read using a μQuant 96-well plate reader and the associated KC4 software (BioTek, Winooski, VT).

Techniques: In Vitro, WST-1 Assay, Expressing, Transfection, Plasmid Preparation, Activity Assay, Concentration Assay

Journal: Foods

Article Title: Olive Pomace Oil versus High Oleic Sunflower Oil and Sunflower Oil: A Comparative Study in Healthy and Cardiovascular Risk Humans

doi: 10.3390/foods11152186

Figure Lengend Snippet: Effects of olive pomace oil (OPO), high oleic sunflower oil (HOSO) and sunflower oil (SO) on endothelial function biomarkers.

Article Snippet: Regarding endothelial function biomarkers, endothelial nitric oxide synthase (eNOS, SEA868Hu), E-selectin (SEA029Hu) and P-selectin (SEA569Hu) plasma concentrations were determined in duplicate by ELISA (Cloud-Clone Kit Corp., Katy, TX, USA) using a Bio-Tek ® Synergy™ HT Multi-Detection Microplate Reader controlled by BioTek ® Gen5 version 2.01.14 software (BioTek Instruments, Winooski, VT, USA).

Techniques:

Journal: Foods

Article Title: Olive Pomace Oil versus High Oleic Sunflower Oil and Sunflower Oil: A Comparative Study in Healthy and Cardiovascular Risk Humans

doi: 10.3390/foods11152186

Figure Lengend Snippet: Effects of olive pomace oil (OPO), high oleic sunflower oil (HOSO) and sunflower oil (SO) on oxidation and antioxidant biomarkers.

Article Snippet: Regarding endothelial function biomarkers, endothelial nitric oxide synthase (eNOS, SEA868Hu), E-selectin (SEA029Hu) and P-selectin (SEA569Hu) plasma concentrations were determined in duplicate by ELISA (Cloud-Clone Kit Corp., Katy, TX, USA) using a Bio-Tek ® Synergy™ HT Multi-Detection Microplate Reader controlled by BioTek ® Gen5 version 2.01.14 software (BioTek Instruments, Winooski, VT, USA).

Techniques:

Journal: Foods

Article Title: Olive Pomace Oil versus High Oleic Sunflower Oil and Sunflower Oil: A Comparative Study in Healthy and Cardiovascular Risk Humans

doi: 10.3390/foods11152186

Figure Lengend Snippet: Effects of olive pomace oil (OPO), high oleic sunflower oil (HOSO) and sunflower oil (SO) on endothelial function biomarkers.

Article Snippet: Regarding endothelial function biomarkers, endothelial nitric oxide synthase (eNOS, SEA868Hu), E-selectin (SEA029Hu) and P-selectin (SEA569Hu) plasma concentrations were determined in duplicate by ELISA (Cloud-Clone Kit Corp., Katy, TX, USA) using a Bio-Tek ® Synergy™ HT Multi-Detection Microplate Reader controlled by BioTek ® Gen5 version 2.01.14 software (BioTek Instruments, Winooski, VT, USA).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: The chromatin-binding protein Smyd1 restricts adult mammalian heart growth

doi: 10.1152/ajpheart.00235.2016

Figure Lengend Snippet: Transcriptome analyses reveal Smyd1 to be a transcriptional repressor of a core set of developmental genes. Transcriptome analyses were performed separately on right ventricles (RV) and left ventricles (LV) from mice with deletion of Smyd1 at 2 wk and 9 wk after removal from tamoxifen diet (controls were normal diet-fed littermates). A: all genes measured in both groups, RV and LV, are displayed in heat map format (red is upregulation, green downregulation, and black statistically unchanged) and clustered according to similar behavior. 2 clusters were defined as indicated, and the functionality of genes in those clusters was determined by gene ontology (GO) analysis. The networks for cluster 1 (B) and 2 (C) for the biological process ontology are shown. In the network figures, node size indicates the P value, and linkage between two terms indicates the relatedness, as determined by κ statistics. The color indicates functional groups with each group represented by their most significant leading term. All terms shown in the network image of cluster 1 are filtered at P < 0.005; those in cluster 2 are filtered at P < 0.0001. Bioinformatic analyses of transcripts with altered expression in the LV, at week 2 (D) and week 9 (E) after Tmx treatment, show significant enrichment in genes involved in extracellular matrix remodeling, fibrosis, and transcriptional repression. MF, molecular function; BP = biological process; CC, cellular component; KEGG, KEGG analysis; ITP, Interpro analysis. Microarray results for several of these transcripts were subsequently validated by RT-PCR (F: all those we attempted to validate are shown; n = 3–6/group; *P < 0.05 vs no tamoxifen group). Chromatin immunoprecipitation (ChIP) and qPCR for Smyd1 (using FLAG antibody) show enrichment in the promoter region of target genes tgfbeta3 (G) and nppa (H); however, no corresponding enrichment of histone H3 lysine K4 trimethylation was detected in these regions (bottom). *P ≤ 0.05. I: luciferase reporter assay using the tgfbeta3 and nppa promoters confirms that Smyd1 acts as a transcriptional repressor by inhibiting transcription of these genes; n = 6/group; *P < 0.05. J: as a negative control, Smyd1 is enriched by ChIP-PCR at neither β-tubulin nor β-actin using primers shown previously to target the regulatory regions upstream of these genes [−3 kb for β-tubulin (24), −73 bp for β-actin (29)].

Article Snippet: After 24 h, the cells were transfected with 75 ng of human TGF-β3, Nppa, negative control (scrambled), or positive control (actin) luciferase reporter construct (SwitchGear Genomics) using FuGene HD (SwitchGear Genomics) at a 6:1 ratio to DNA and incubated for 48 h. Luciferase activity was assayed using the LightSwitch Luciferase assay reagent (SwitchGear Genomics) according to the manufacturer's instructions and measured using a BioTek Synergy Neo HTS Multi-Mode microplate reader.

Techniques: Functional Assay, Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Negative Control