Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Representative RNA dot-blot illustrating 8-oxoGua levels. Total RNA from virocells (MOI = 1, 24 hpi) or treatment with NAC (10 mM) was serially diluted twofold and probed with an anti-8-oxoGua antibody. (B) Anti-8-oxoGua antibody pull down from total RNA extracted from virocells (MOI = 1). Levels of RSV gRNA in immunoprecipitants were quantified by qRT-PCR. (C) Immunoprecipitation using anti-8-oxoGua antibody from total RNA extracted from virocells (MOI = 1, 24 hpi), with treatments of NAC (10 mM) or EUK-8 (100 μM) initiated post-inoculum. Levels of RSV gRNA in immunoprecipitants were determined by qRT-PCR. (D) Quantification of 8-oxoGua levels in RSV G and L mRNA by qRT-PCR. Total RNA was extracted from virocells (MOI = 1, 24 hpi), mRNA was enriched using Oligo dT beads, and incubated with 8-oxoGua or negative control antibody. In B-D, Fold change was calculated by 2^- [Ct (test Ab)-Ct (negative Ab)]. (E) Quantification of 8-oxoGua and m6A levels by LC_MS/MS. Left panel: Strategy for extracting viral RNA from RSV virions or virocells (MOI = 1, 24 hpi). Treatment with TH5487 (10 μM) was initiated post inoculum. RSV virions were purified by sucrose ultracentrifugation from cell culture supernatant. Biotin-labeled RSV antibody was used to pull down N protein-associated RNA from virocells without crosslinking. Right panel: Graphical representation of 8-oxoGua level in virions (#1) and N protein-associated RNA from virocells (#2 and #3, upper right panel). m6A levels were exclusively assessed in virions (#1, lower right panel). Data are expressed as the average peak area per ng RNA. n = 3 biological replicates. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. Significance levels are indicated as * p <0.05, ** p < 0.01, *** p < 0.001. Fig 2E created with Biorender.com .

Article Snippet: TH5487 (SelleckChem; 30 mg/ kg) in 200 μL of solvent (5% DMSO, 10% Tween 80 in saline) was administered via the peritoneal route every 8 h after RSV challenge.

Techniques: Dot Blot, Quantitative RT-PCR, Immunoprecipitation, Incubation, Negative Control, Liquid Chromatography with Mass Spectroscopy, Purification, Cell Culture, Labeling

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) OGG1 binds RNA containing 8-oxoGua. The secondary structure of the undenatured single-strand probe was predicted using software as described in Materials and Methods. The probe contains a single 8-oxoGua (marked in red) at the end of GGGG. EMSA was performed using single-stranded RNA (8oxo-rG), either denatured at 95°C for 5 min (Lanes 1–4) or undenatured (Lanes 5–8). Additionally, EMSA was conducted with the double-stranded RNA probe (8oxo-rG: rC), where 8oxo-rG anneals with its complementary genome sequence (rC) (Lanes 9–12). (B) Representative EMSA image demonstrating OGG1’s binding affinity for annealed RNA containing 8-oxoGua. (C) Representative image showing OGG1’s excision activity on 8-oxoGua within DNA, contrasting with its inactivity towards RNA. (D) EMSA visualization indicating that TH5487 impedes OGG1’s binding to RNA harboring 8-oxoGua. (E) EMSA depiction showing OGG1’s interaction with a DNA-RNA hybrid that includes the gene-start (GS) motif. Total RNA (1 μg) extracted from virocells (MOI = 1, 24 hpi) was hybridized with Cy5-labelled DNA probes (20 nM) containing the intergenic region (IG), the gene-start (GS), and the gene-end (GE) of G gene. Following incubation with recombinant OGG1 (100 nM), the assay proceeded to EMSA. Oligo sequences for EMSA and OGG1 glycosylase activity are listed in . n = 3 biological replicates.

Article Snippet: TH5487 (SelleckChem; 30 mg/ kg) in 200 μL of solvent (5% DMSO, 10% Tween 80 in saline) was administered via the peritoneal route every 8 h after RSV challenge.

Techniques: Software, Sequencing, Binding Assay, Activity Assay, Incubation, Recombinant

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Left panel: Strategy for enrichment of viral RNAs interacting with OGG1 and N protein from virocells after formaldehyde crosslinking. Treatment with TH5487 (10 μM) was initiated post inoculum (MOI = 1, 24 hpi). RNA-immunoprecipitation (RIP) was carried out using antibodies against OGG1 or RSV N protein. Right panel: Quantification of RSV genomic RNA levels after antibody pull-down, determined by qRT-PCR. Fold change was calculated by 2^- [Ct (test Ab)-Ct (negative Ab)]. n = 3 biological replicates. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. *** p <0.001. (B) OGG1 RIP-Seq analysis in virocells (MOI = 1, 24 hpi) illustrating the distribution of RSV-specific reads across two independent experiments (visualized in blue and red tracks). Data are presented as the log2 ratio of reads obtained from anti-OGG1 immunoprecipitation relative to input RNA, mapped against the RSV genome (GenBank: M74568.1). Peaks were called positive if the log2 enrichment score was ≥1. (C) Annotation and categorization of cellular RNA reads obtained from OGG1 RIP-Seq aligned to the human genome HG38 (GenBank: GCA_000001405.29), presented as the percentage of OGG1-RIP peaks. UTR, untranslated region of mRNAs; CDS, exon regions that code for protein; und., undefined. Fig 4A created with Biorender.com .

Article Snippet: TH5487 (SelleckChem; 30 mg/ kg) in 200 μL of solvent (5% DMSO, 10% Tween 80 in saline) was administered via the peritoneal route every 8 h after RSV challenge.

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A-B) hSAECs were infected with RSV (MOI = 0.1) and viral titers were assessed 3 days post-infection in scenarios where OGG1 expression was either (A) down-regulated via siRNA or (B) completely eliminated by CRISPR/Cas9 knockout. NT, non-targeting siRNA. The knockout was overcompensated by ectopic expression of OGG1. The lower panel displays immunoblot analysis of OGG1 expression in cell lysates, with Actin serving as a loading control. (C) Following 1-hour post inoculation (MOI = 0.1), hSAECs were treated with TH5487, TH2840, or O8, and viral titers were determined by plaque assays (3 dpi). In D-I, hSAECs were infected with RSV (MOI = 0.5), and total RNA was extracted first at indicated times post-infection. Experiments include OGG1 knockout by CRISPR/Cas9 in hSAECs (D and G), down regulation of OGG1 expression by siRNA (E and H), and inhibition of OGG1’s reading function by TH5487 (F and I). (D-F) mRNA was isolated using Oligo dT beads, and G mRNA level was quantified by RT-qPCR. (G-I) After mRNA isolation, RSV gRNA levels were determined via RT-qPCR. n = 3. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. Significance levels are indicated as * p <0.05, ** p < 0.01, *** p < 0.001. (J) Efficiency of primer extension on RNA template containing 8-oxoGua. Moloney murine leukemia virus reverse transcriptase (RT) was used in the assay. H 2 O 2 , 800 μM. OGG1, 50 nM. P indicates primer. E indicates extension.

Article Snippet: TH5487 (SelleckChem; 30 mg/ kg) in 200 μL of solvent (5% DMSO, 10% Tween 80 in saline) was administered via the peritoneal route every 8 h after RSV challenge.

Techniques: Infection, Expressing, CRISPR, Knock-Out, Western Blot, Control, Inhibition, Isolation, Quantitative RT-PCR, Virus, Reverse Transcription

Journal: Journal of Extracellular Vesicles

Article Title: The 8‐oxoguanine DNA glycosylase‐synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress

doi: 10.1002/jev2.12505

Figure Lengend Snippet: SYT7 is a potential target gene of OGG1. (A, B) After A549 cells were transfected with scrambled or OGG1 siRNA for 48 h, the mRNA levels of OGG1 (A) and SYT7 (B) were examined. (C) Cells were treated as panel (A). Western blotting was used to determine the protein levels of OGG1 and SYT7. Right, quantification of OGG1 and SYT7. (D, E) The mRNA (D) and protein (E) levels of SYT7 in the WT and OGG1‐KO cells were examined by RT‐qPCR and Western blotting. (F) Analysis of CpG islands 2 kb upstream of the TSS region of SYT7. Upper graph: A plot of the ratio of observed GC content to expected GC content. Middle graph: GC content percentage. Lower graph: The CpG island was predicted to exist at −844 to −55 nucleotides (nt) upstream of the TSS region of SYT7. (G, H) The mRNA (G) and protein (H) levels of SYT7 in A549 cells treated with or without 20 µM H 2 O 2 were analysed. Right, quantification of SYT7 level. (I, J) After cells were treated with 20 µM H 2 O 2 and 10 mM NAC for 24 h, the mRNA (I) and protein (J) levels of SYT7 were analysed. Right, quantification of SYT7. (K, L) After cells were treated with or without 10 µM Th5487 for 24 h, the mRNA (K) and protein (L) levels of SYT7 were analysed. Right, quantification of SYT7 level. (M, N) After cells were treated with or without 10 µM OGG1‐IN‐08 for 24 h, the mRNA (M) and protein (N) levels of SYT7 were analysed. Right, quantification of SYT7 level. All of the data are expressed as mean values ± SEM ( n = 3); ** p < 0.01, *** p < 0.001. (Student's t ‐test).

Article Snippet: In the Th5487 treatment experiment, after 10 days of xenograft, BALB/c‐Nude mice (GemPharmatech Co., Ltd., Nanjing, China) received daily intraperitoneal injections of Th5487 (30 mg/kg, 200 µL) daily.

Techniques: Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Extracellular Vesicles

Article Title: The 8‐oxoguanine DNA glycosylase‐synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress

doi: 10.1002/jev2.12505

Figure Lengend Snippet: OGG1 promotes the binding of NF‐κB to target sites on the SYT7 promoter. (A) Analysis of the SYT7 promoter. NF‐κB binding sites in the SYT7 promoter region were predicted by JASPAR database. (B) The NF‐κB level in the cytoplasm and nucleus was analysed by Western blotting. Right, quantification of NF‐κB level. (C, D) A549 cells were exposed to 20 ng/mL TNF‐α for 4 h, and the mRNA (C) and protein (D) levels were analysed. Right, quantification of SYT7 level. (E, F) Cells were treated with or without 10 µM JSH‐23, and the mRNA (E) and protein (F) levels of SYT7 were analysed. Right, quantification of SYT7 level. (G, H) Cells were treated with or without 10 µM JSH‐23 or/and 10 µM OGG1‐IN‐08. The mRNA (G) and protein (H) levels of SYT7 were analysed. Right, quantification of SYT7 level. (I, J) 8‐oxoG increased the occupancy of OGG1 (I) or NF‐κB (J) on the SYT7 promoter. Purified OGG1 protein or nucleoprotein was subjected to EMSA with different FAM‐labelled probes. The arrow indicated the position of the NF‐κB or OGG1‐probe complex. (K) Occupancy of NF‐κB on 8‐oxoG‐containing DNA in nuclear extracts. Nuclear extracts (50 µg per sample) were incubated with G or 8‐oxoG probes for 12 h, and the protein‐DNA complexes were analysed by Western blotting. (L, M) ChIP‐qPCR assay determined the H 2 O 2 ‐induced enrichment of OGG1 (L) or NF‐κB (M) on the SYT7 promoter. (N) ChIP‐qPCR detected the enrichment of NF‐κB at the SYT7 promoter in WT or OGG1‐KO cells under oxidative stress. (O) EMSA detected the effect of OGG1‐IN‐08 and Th5487 exposure on OGG1 binding to oxidised DNA strands. 10 µM OGG1‐IN‐08 or 10 µM Th5487 was incubated with 100 ng OGG1. (P) ChIP‐qPCR analysis of NF‐κB enrichment at the SYT7 promoter under oxidative stress with or without Th5487 treatment. (Q) ChIP‐qPCR analysis of NF‐κB enrichment at the SYT7 promoter under oxidative stress with or without OGG1‐IN‐08 treatment. All of the data are expressed as mean values ± SEM ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001. (Student's t ‐test).

Article Snippet: In the Th5487 treatment experiment, after 10 days of xenograft, BALB/c‐Nude mice (GemPharmatech Co., Ltd., Nanjing, China) received daily intraperitoneal injections of Th5487 (30 mg/kg, 200 µL) daily.

Techniques: Binding Assay, Western Blot, Purification, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: The 8‐oxoguanine DNA glycosylase‐synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress

doi: 10.1002/jev2.12505

Figure Lengend Snippet: Overexpression of SYT7 partially restores the down‐regulation of migration caused by OGG1 deletion. (A) After overexpressing SYT7 in cells knocked down OGG1, transwell assay was used to detect migrated cells. Right, quantification of migrated cells. (B) Cells were treated as panel (A). Protein levels of EMT markers were verified by western blot assays. Right, quantitative analysis. (C) After overexpressing SYT7 in cells knocked out OGG1, transwell assay was used to detect migrated cells. Right, quantification of migrated cells. (D) Cells were treated as panel (C). Protein levels of EMT markers were verified by western blot assays. Right, quantitative analysis. (E) The total protein concentration of isolated EVs was determined by BCA assay. (F) Western blotting analysed equal amounts of cell‐isolated EVs for markers content. Right, quantitative analysis of markers. (G) The content of E‐cadherin in equal amounts of EVs was quantified by Western blotting. (H) Transwell assay detected the invasion and migration ability of A549 cells after Th5487 exposure. Right, quantification of migrated cells. (I) Cells were treated with Th5487 for 24 h. EMT‐related markers were detected by Western blotting. Right, quantification of the marks. (J) After cells were treated with Th5487 for 24 h, EVs from the culture supernatant were isolated by ultra‐fractionation. The total protein concentration of isolated EVs was determined by BCA assay. (K) ELISA determined the content of CD63 in isolated EVs. (L) Western blotting analysed equal amounts of cell‐isolated EVs for markers content. Right, quantitative analysis of markers. (M) The content of E‐cadherin in equal amounts of EVs was quantified by Western blotting. All of the data are expressed as mean values ± SEM ( n = 3); ** p < 0.01, *** p < 0.001. (Student's t ‐test).

Article Snippet: In the Th5487 treatment experiment, after 10 days of xenograft, BALB/c‐Nude mice (GemPharmatech Co., Ltd., Nanjing, China) received daily intraperitoneal injections of Th5487 (30 mg/kg, 200 µL) daily.

Techniques: Over Expression, Migration, Transwell Assay, Western Blot, Protein Concentration, Isolation, BIA-KA, Fractionation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: The 8‐oxoguanine DNA glycosylase‐synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress

doi: 10.1002/jev2.12505

Figure Lengend Snippet: OGG1 deletion reduces lung cancer metastasis in vivo. (A) Schematic diagram of the experimental process of zebrafish xenograft. CD‐Dil‐labelled A549 cells (red) were injected into the perivitelline space of each zebrafish. (B) A zebrafish xenograft model was used to evaluate the metastatic ability of A549 cells after OGG1 knockout. Representative images of zebrafish were shown. (C) Schematic diagram of establishing a lung cancer metastasis model by injecting A549 cells into the tail vein. (D) In vivo bioluminescence imaging of mice 40 days after cell injection. (E) Gross anatomy of mouse lungs. (F) Representative HE staining images of lungs from each group. Scale bar: 200 µm. (G, H) Zebrafish juvenile survival rate (G) and heart rate (H) at 120 hpf. Conventional one‐way ANOVA test was performed considering the experimental group as the independent variable. ns stands for no significance. (I) Schematic diagram of an experimental zebrafish xenograft experiment using A549 cells to test the anti‐tumour effect of Th5487. (J) Zebrafish xenograft model was used to evaluate the effect of Th5487 on A549 cell metastasis. Representative images of zebrafish were shown. (K) A mouse lung metastasis model was established by injecting A549 cells into the tail vein. Ten days after cell injection, mice were intraperitoneally injected with 30 mg/kg Th5487 or a corresponding volume of PBS every 2 days. (L) Gross anatomy of mouse liver. (M) Representative HE staining images of lungs and liver in each group. Scale bar: 50 µm. (N) Representative images from IHC staining for SYT7. Scale bar: 50 µm. All of the data except zebrafish juvenile survival rate and heart rate are expressed as mean values ± SEM ( n = 3); ** p < 0.01, *** p < 0.001. (Student's t ‐test).

Article Snippet: In the Th5487 treatment experiment, after 10 days of xenograft, BALB/c‐Nude mice (GemPharmatech Co., Ltd., Nanjing, China) received daily intraperitoneal injections of Th5487 (30 mg/kg, 200 µL) daily.

Techniques: In Vivo, Injection, Knock-Out, Imaging, Staining, Immunohistochemistry