Journal: Genetics

Article Title: A novel role for the E2F transcription factor and the ER stress sensor IRE1 in cytoplasmic DNA accumulation

doi: 10.1093/genetics/iyaf190

Figure Lengend Snippet: DNA accumulates in the cytoplasm of de2f1b SGs. a) Control ( w 1118 ) and de2f1b SGs ( de2f1b/Df ) are stained with an antibody against double-stranded DNA (anti-dsDNA) and DAPI. Lower panels show magnified views of the box area (scale bars: 50 and 20 μm for magnified images). Arrowheads mark anti-dsDNA signals that overlap with cytoplasmic DAPI. b) de2f1b SGs expressing mitochondrially localized GFP (Mito-GFP) are stained with anti-dsDNA. Arrowheads mark anti-dsDNA signals that overlap with cytoplasmic DAPI and asterisks indicate anti-dsDNA signals that demark mitochondria (scale bars: 10 μm) (c) Antimicrobial peptide gene (AMP) expression levels were measured by RT-qPCR. Relative fold difference of indicated AMPs between control and de2f1b SGs are shown (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05). d) The relative fold differences of the expression of previously identified STING-regulated genes between control and de2f1b SGs is determined by RT-qPCR (**** P < 0.0001).

Article Snippet: The anti- γ H2Av (UNC93-5.2.1) and anti-double-stranded dsDNA (autoanti-dsDNA) antibodies were obtained from the Developmental Studies Hybridoma Bank ( https://dshb.biology.uiowa.edu ).

Techniques: Control, Staining, Expressing, Quantitative RT-PCR

Journal: Genetics

Article Title: A novel role for the E2F transcription factor and the ER stress sensor IRE1 in cytoplasmic DNA accumulation

doi: 10.1093/genetics/iyaf190

Figure Lengend Snippet: The endoplasmic reticulum (ER) homeostasis is deregulated in de2f1b SGs. a) RNA-seq was performed to compare the gene expression profile between control and de2f1b SGs. Volcano plot of differentially expressed genes in de2f1b SGs is shown. Each point represents a gene plotted by log 2 fold change (x-axis) and –log 10 adjusted P -value ( y -axis). Significantly upregulated and significantly downregulated genes are shown (genes with adjusted P -value < 0.05). Five genes that belongs to the category “protein processing in the ER” are highlighted and labeled. b) Ontology analysis was performed with genes whose expressions are downregulated in de2f1b SGs. The top 5 biological processes identified from the ontology analysis are shown. The intensity of the color depicts the false discovery rate, and the size of the circles depicts the fold enrichment. c) A GFP construct tagged with ER localization and retention signals is used to visualize ER morphology (ER:GFP). Anti-dsDNA was also used to determine the abundance of cytoDNA. Magnified view of the boxed area is also shown (scale bars: 50 and 25 μm for magnified images). (d) A genomic construct, in which the coding region of the SGS3 gene is fused with the GFP sequence (SGS:GFP), is used to monitor the expression of “glue proteins” in control and de2f1b SGs. Anti-dsDNA was also used to determine the abundance of cytoDNA. White arrowheads point to cells with a high level of cytoDNA (scale bars: 50 and 25 μm for magnified images). FB: Fat body.

Article Snippet: The anti- γ H2Av (UNC93-5.2.1) and anti-double-stranded dsDNA (autoanti-dsDNA) antibodies were obtained from the Developmental Studies Hybridoma Bank ( https://dshb.biology.uiowa.edu ).

Techniques: RNA Sequencing, Gene Expression, Control, Labeling, Construct, Sequencing, Expressing

Journal: Genetics

Article Title: A novel role for the E2F transcription factor and the ER stress sensor IRE1 in cytoplasmic DNA accumulation

doi: 10.1093/genetics/iyaf190

Figure Lengend Snippet: IRE-1 is required for proper ER development and preventing cytoDNA accumulation. a) Ire1 was depleted in the SG and their effect on the ER was visualized by ER-localized GFP (ER:GFP). A control SG ( sg-G4 ) and a representative image of ire1 -depleted SGs are shown ( sg-G4 > ire1 RNAi ). b) Xbp1 was depleted in the SG ( sg-G4 > xbp1 RNAi ) and their effect on the ER network was visualized. c) Ire1 was depleted in the SG ( sg-G4 > ire1 RNAi ) and cytoDNA was visualized by anti-dsDNA. d) The SGS:GFP genomic construct was used to monitor the expression of glue proteins in control and ire-1 depleted SGs at 110–120 h AEL. cytoDNA was also visualized by anti-dsDNA (scale bars for all the images: 50 and 20 μm for magnified images).

Article Snippet: The anti- γ H2Av (UNC93-5.2.1) and anti-double-stranded dsDNA (autoanti-dsDNA) antibodies were obtained from the Developmental Studies Hybridoma Bank ( https://dshb.biology.uiowa.edu ).

Techniques: Control, Construct, Expressing

Journal: Genetics

Article Title: A novel role for the E2F transcription factor and the ER stress sensor IRE1 in cytoplasmic DNA accumulation

doi: 10.1093/genetics/iyaf190

Figure Lengend Snippet: IRE1 overexpression differently affects the ER in de2f1b SGs. a) XBP1 or IRE1 was overexpressed in control ( sg-G4 ) and de2f1b SGs ( sg-G4; de2f1b/Df ), and the ER was visualized by ER:GFP. Magnified images of the boxed area are shown in the lower panels. b) The effect of overexpressing XBP1 or IRE1 on cytoDNA accumulation in wild-type SGs is determined. The presence of cytoDNA was visualized by anti-dsDNA. Arrowheads point to cytoDNA. Magnified images of the boxed area are shown in the lower panels (scale bars for all the images: 50 and 20 μm for magnified images).

Article Snippet: The anti- γ H2Av (UNC93-5.2.1) and anti-double-stranded dsDNA (autoanti-dsDNA) antibodies were obtained from the Developmental Studies Hybridoma Bank ( https://dshb.biology.uiowa.edu ).

Techniques: Over Expression, Control

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