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: Arthritis Research & Therapy

Article Title: Early treatment with hydroxychloroquine prevents the development of endothelial dysfunction in a murine model of systemic lupus erythematosus

doi: 10.1186/s13075-015-0790-3

Figure Lengend Snippet: Anti–double-stranded DNA (anti-dsDNA) levels at different time points. The results are expressed as optical density (OD) (mean of the OD readings of serum at 450 nm) as measured using a microtiter plate reader (Ultrospec 2000; Amersham Pharmacia Biotech, Little Chalfont, UK). * P = 0.02 in NZB/W F1 mice treated with hydroxychloroquine (NZ-HCQ) vs. NZB/W F1 (NZ) mice at 24 weeks

Article Snippet: Serum titers of anti–double-stranded DNA (anti-dsDNA) autoantibodies were analyzed with a commercial enzyme-linked immunosorbent assay kit (Alpha Diagnostic International, San Antonio, TX, USA), 24-h urinary protein excretion was collected in metabolic cages and measured by dipstick analysis (Bayer, Leverkusen, Germany).

Techniques: