Journal: Journal of Virological Methods

Article Title: Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

doi: 10.1016/j.jviromet.2014.12.002

Figure Lengend Snippet: Summary of viruses contained in the multi-viral mix.

Article Snippet: The DNA input for Nextera library preparations was either the RT and Klenow DNA polymerase dsDNA products or that same product following PCR amplification products ( ).

Techniques: Cell Culture, Virus

Journal: Antioxidants & Redox Signaling

Article Title: Inhibition of NFE2L2-Antioxidant Response Element Pathway by Mitochondrial Reactive Oxygen Species Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease

doi: 10.1089/ars.2016.6831

Figure Lengend Snippet: NFE2L2 transcriptional activation in chagasic myocardium (±MnSOD). Mice were infected with T. cruzi and harvested at 150 days pi. (A) Western blotting. Heart tissue homogenates (A.a, A.c) and nuclear fractions (A.b, A.d) were subjected to Western blotting with anti-NFE2L2 antibody (A.a, A.b), and densitometry analysis of the NFE2L2 signal (A.c, A.d) is presented. (B) Western blotting and densitometry analyses of Keap1 (B.a, B.b) and HO1 (B.a, B.c) levels in heart homogenates. Densitometry analysis of WB bands in heart homogenates and nuclear fractions were normalized to GAPDH and Lamin A/C, respectively. (C) NFE2L2–ARE binding. NFE2L2 double-stranded (ds) DNA-binding capacity in nuclear fractions of heart tissue from WT and MnSODtg mice (±Tc) was measured as described in Materials and Methods section. (D) RT-qPCR analysis for γGCS mRNA level in WT and MnSODtg mice (±Tc). Data are plotted as mean value ± SEM (n = 6–10 mice per group). Significance is shown as ***,###p < 0.001. ARE, antioxidant response element; γGCS, gamma-glutamyl cysteine synthase; HO1, heme oxygenase-1; Keap1, Kelch-like ECH-associated protein 1; mRNA, messenger RNA; NFE2L2, nuclear factor (erythroid 2)-like 2; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: NFE2L2-double-stranded DNA binding assay The nuclear fractions (5 μg) from the heart tissue of WT and MnSOD tg mice (normal and Tc infected) were added in triplicate to the 96-well plate precoated with double-stranded DNA (dsDNA) sequence containing the NFE2L2 response element (Cat. No. 600590; Cayman Chemicals).

Techniques: Activation Assay, Infection, Western Blot, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Antioxidants & Redox Signaling

Article Title: Inhibition of NFE2L2-Antioxidant Response Element Pathway by Mitochondrial Reactive Oxygen Species Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease

doi: 10.1089/ars.2016.6831

Figure Lengend Snippet: MnSOD preserves NFE2L2–ARE functional activity in Tc-infected cells. (A) Confocal microscopy. Cardiac myocytes were transfected with empty vector (A.a–A.f) or pBI.EGFP–MnSOD (A.g–A.l) and incubated with (A.d–A.f, A.j–A.l) or without (A.a–A.c, A.g–A.i) T. cruzi for 24 h. Cells were stained with DAPI (nuclear blue, A.a, A.d, A.g, A.j) and Alexa Fluor 568-conjugated anti-NFE2L2 antibody (red, A.b, A.e, A.h, A.k) and analyzed by confocal fluorescence microscopy. Overlay images (A.c, A.f, A.i, A.l) show cytosolic and nuclear localization of NFE2L2. (B) Antioxidant genes' expression. RT-qPCR measurement of mRNA levels for (B.a) NFE2L2, (B.b) GCLM, (B.c) Trx, (B.d) GST, and (B.e) NQO1 in cardiomyocytes transfected with MnSOD expression plasmid and infected with T. cruzi for 24 h. Fold change was determined after normalizing the data with GAPDH mRNA. (C) NFE2L2 transcriptional activity. HeLa cells were transiently transfected with pGL3-promo-2x-hHO1 (C.a), pGL3-promo-2x-hNQO1 (C.b), or pGL3-promo-2x-hGCLM (C.c) plasmids consisting of luciferase encoding gene under NFE2L2-binding ARE sequence from HO1, NQO1, and GCLM, respectively. Cells were cotransfected with an MnSOD-expressing plasmid and a Renilla luciferase plasmid (positive control for normalization of transfection efficiency). Transfected cells were infected (cell:Tcratio, 1:5) for 24 h. The relative NFE2L2 transcriptional activity for HO1, NQO1, and GCLM was measured by using a dual luciferase assay and normalized to Renilla luciferase activity. Data (mean ± SEM) are representative of three independent experiments (triplicate observations per experiment). Significance is shown as *p < 0.05, **,##p < 0.01, ***,###p < 0.001. GCLM, glutamate-cysteine ligase modifier subunit; GST, glutathione S transferase; NQO1, NAD(P)H dehydrogenase, quinone 1; Trx, thioredoxin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: NFE2L2-double-stranded DNA binding assay The nuclear fractions (5 μg) from the heart tissue of WT and MnSOD tg mice (normal and Tc infected) were added in triplicate to the 96-well plate precoated with double-stranded DNA (dsDNA) sequence containing the NFE2L2 response element (Cat. No. 600590; Cayman Chemicals).

Techniques: Functional Assay, Activity Assay, Infection, Confocal Microscopy, Transfection, Plasmid Preparation, Incubation, Staining, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Luciferase, Binding Assay, Sequencing, Positive Control