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: Cells

Article Title: Retinal Neuron Is More Sensitive to Blue Light-Induced Damage than Glia Cell Due to DNA Double-Strand Breaks

doi: 10.3390/cells8010068

Figure Lengend Snippet: Blue light up-regulated Ku80 expression in glia cells. ( A ) Both blue light and white light treatment do not affect the mRNA expression of ligase IV in primary cultured retinal cells. ( B ) Both blue light and white light treatment do not affect the mRNA expression of Mre11 in primary cultured retinal cells. ( C ) The mRNA expression of Ku80 is significantly up-regulated in primary cultured retinal cells upon blue light exposure, compared with control and white light treatment. ( D ) The Ku80 protein expression is significantly up-regulated in primary cultured retinal neurocytes upon blue light exposure, compared with control and white light treatment. While the light treatment does not affect the protein expression of ligase IV and Mre11. ( E ) The relative protein expression of ligase IV and Mre11 are presented as histogram. ( F ) The relative protein expression of Ku80 were presented as a histogram. ( G )The pan-caspase inhibitor, Z-VAD FMK did not affect the γ-H2AX formation in retinal neurocytes c induced by blue light. ( H ) The DNA repair inhibitor, NU7441, further promoted the γ-H2AX foci formation in retinal neurocytes upon blue light treatment. ( I ) The relative protein expression γ-H2AX is presented as a histogram. Error bars represent mean ± SD. Asterisks indicate statistically significant differences between control and experimental samples (* p < 0.05, ** p < 0.01).

Article Snippet: The primary antibodies used were as follows: rabbit anti-Ku80 (1:500, Boster, Wuhan, China) and rabbit anti-γ-H2AX (1:1000, CST, Danvers, MA, USA), Mre11(1:200, Boster, Wuhan, China), Ligase IV (1:1000, Proteintech, Wuhan, China), GAPDH (1:10,000, Proteintech, Wuhan, China).

Techniques: Expressing, Cell Culture, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Quercetin inhibits DNA damage responses to induce apoptosis via SIRT5/PI3K/AKT pathway in non-small cell lung cancer.

doi: 10.1016/j.biopha.2023.115071

Figure Lengend Snippet: Fig. 5. Quercetin inhibited NHEJ and HR pathways phosphorylation. Total proteins lysis and extraction after A549 and H1299 cultured with 0, 12.5, 50, and 200 μM quercetin for 24 h. In NHEJ pathways (A) the expression of p-DNA-PKcsS2056 (B, C), KU70 (D, E) and KU80 (F, G), and in HR pathways (H) the phosphorylation of p- ATRS428 (I, J), p-Chek1S345 (K, L), p-ATMS1981 (M, N) and Chek2T68 (O, P) were detected by western blot in both A549 and H1299 cells. And the results were measured by ImageJ and expressed as protein expression relative to GAPDH (mean ± S.D., n = 3). #p > 0.05, *p < 0.05, * *p < 0.01, * **p < 0.001 relative to values in the respective 0 μM group (B, p = 0.0048, R2 =0.7844, F=9.700; C, p = 0.0002, R2 =0.9042, F=25.17; D, p = 0.0054, R2 =0.7782, F=9.358; E, p = 0.0045, R2 =0.7879, F=9.908; F, p = 0.0037, R2 =0.7990, F=10.60; G, p = 0.0016, R2 =0.8379, F=13.79; I, p = 0.0034, R2 =0.8028, F=10.85; J, p = 0.0032, R2 =0.8066, F=11.12; K, p = 0.0010, R2 =0.8564, F=15.91; L, p = 0.0016, R2 =0.8379, F=13.79; M, p = 0.0044, R2 =0.7898, F=10.02; N, p = 0.0065, R2 =0.7676, F=8.806; O, p = 0.0030, R2 =0.0.8087, F=11.27; P, p = 0.0026, R2 =0.8166, F=11.87), One-way ANOVA test. The mRNA in A549 (Q) and H1299 (R) cells was extracted and reverse transcribed to cDNA for RT-qPCR analysis (mean ± S.D., n = 3). #p > 0.05, *p < 0.05, * *p < 0.01, * **p < 0.001, * ** *p < 0.0001 relative to values in the respective 0 μM group, One-way ANOVA test.

Article Snippet: Antibodies to γ-H2AXS139, p-ATRS428 (AP0676) and p-CDK1T161 (AP0324) were from Abclonal Technology (Abclonal, Wuhan, China); antibodies to KU70 (AF0300) and p-Chek1Ser345 (AF3008) were from Affinity Biosciences (Affinity, Jiangsu, China); antibodies to KU80 (PB9464) and p-DNAPKcsS2056 (BM4058) were from Boster Biological Technology (Boster, Wuhan, China); antibodies to p-ATMSER1981 (bsm-54103R) and pChek2Thr68 (ba-3721R) were from Biosynthesis Biotechnology (Bioss, Beijing, China); antibodies to p-PI3K (17366) and p-AKT (4060) were from Cell Signaling Technology (Danvers, MA, USA); antibodies to Caspase-3 (ab32042), Bax (ab32503), Bcl-2 (ab32124), SIRT5 (ab259967), GAPDH (ab9485) and β-tubulin (ab179511) were from Abcam (Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Lysis, Extraction, Cell Culture, Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR