Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: Effects of quercetin against TGEV infection and target network-based mechanistic insights. Unless otherwise specified, IPEC-J2 cells were pretreated with quercetin (80 μM) for 1 h before infection with TGEV (MOI = 1) for 24 h. (A) Flow cytometric analysis of intracellular TGEV-N protein in IPEC-J2 cells infected with TGEV for 24 h. The virus was pre-incubated with or without quercetin (80 μM) at 4 °C for 24 h prior to cell infection (n = 3). (B) Immunofluorescence staining and quantification of intracellular TGEV-N protein (green) and nuclei (DAPI, blue) in IPEC-J2 cells treated under standard quercetin pretreatment conditions (n = 3). Scale bar = 100 μm. (C) Quantitative RT-PCR analysis of TGEV-N mRNA levels in IPEC-J2 cells following quercetin treatment and TGEV infection (n = 4). (D) Live/dead staining using Calcein-AM (green) and propidium iodide (PI, red) in IPEC-J2 cells (n = 3). Scale bar = 100 μm. (E) Volcano plot showing differentially expressed genes (DEGs) in TGEV-infected intestinal organoids from GEO dataset GSE182240 (F) Venn diagram illustrating 82 overlapping genes among TGEV-related genes (GeneCards), quercetin targets (TCMSP), and DEGs from GSE182240 . (G) Protein-protein interaction (PPI) network of the overlapping genes constructed using the STRING database. (H) Compound-target-pathway network visualizing quercetin and its putative host targets in the context of TGEV infection. (I) KEGG pathway enrichment analysis of the overlapping genes. (J) Gene Set Enrichment Analysis (GSEA) of transcriptomic data ( GSE182240 ) showing enrichment of inflammatory signaling pathways including NF-κB, TNF, and JAK-STAT pathways in TGEV-infected organoids (K) GO cellular component enrichment. (L) GSEA analysis indicating suppression of mitochondrial protein-containing complex-related genes in TGEV-infected organoids. (M) Chord diagram depicting associations between GO terms and core target genes. Data were expressed as mean ±SEM. ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: Infection, Virus, Incubation, Immunofluorescence, Staining, Quantitative RT-PCR, Construct, Protein-Protein interactions

Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: TGEV infection induces mitochondrial dysfunction and inflammatory signaling activation. IPEC-J2 cells were infected with TGEV (MOI = 1) for 0, 6, 12, and 24 hours to assess time-dependent changes in inflammation and mitochondrial function. (A) Western blot analysis of phosphorylated and total levels of STAT1, STAT3, IκBα, and NF-κB p65 at indicated time points post-infection. (B) Quantitative RT-PCR analysis of mRNA expression of pro-inflammatory cytokines ( IL-1β , IL-6 , IL-18 , TNF-α ), interferon-stimulated genes ( IFNB1 , CXCL10 ), and NLRP3 at 24 hours post-infection. (n = 3-4) (C) Immunofluorescence staining of NF-κB p65 (green) and DAPI (blue) showing subcellular distribution of NF-κB p65 in control and TGEV-infected cells. Quantification of nuclear/cytoplasmic ratio is shown on the right. Scale bar = 50 μm. (D-E) Western blot analysis of mitochondrial electron transport chain complexes I-V at various time points post-infection. (F) Quantification of relative mtDNA copy number by qPCR following TGEV infection (n = 3-4). (G) Immunofluorescence staining of mtROS (red) and nuclei (DAPI, blue) in control and TGEV-infected cells. Scale bar = 50 μm. (H) Flow cytometric analysis of mitochondrial health indicators, including mitochondrial membrane potential, mtROS, and mitochondrial biomass at various time points (3, 6, 12, and 24 h) following infection (n = 3-4). Data were expressed as mean ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: Infection, Activation Assay, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Control, Membrane

Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: Quercetin alleviates TGEV-induced inflammation and mitochondrial damage. IPEC-J2 cells were pretreated with quercetin (80 μM) for 1 h before infection with TGEV (MOI = 1) for 24 h. (A) Western blot analysis of phosphorylated and total levels of IκBα and NF-κB p65 in each group. (B) Quantitative RT-PCR analysis of mRNA expression of TNF-α , IL-6 , IL-1β , NLRP3 , IFNB1 , and CXCL10 (n = 3-4). (C) Immunofluorescence staining of NF-κB p65 (green) and nuclei (DAPI, blue) to assess p65 nuclear translocation. Scale bar = 50 μm. (D) Molecular docking analysis of quercetin with three inflammation-related proteins (NFκBIA, TNFA, STAT1). (E) Western blot analysis of mitochondrial electron transport chain complexes I-V. (F) Quantification of mtDNA copy number by qPCR in each group (n = 3). (G) Flow cytometric analysis of MMP, mitochondrial biomass, and mtROS (n = 3). (H) Immunofluorescence staining of mtROS (red) and nuclei (DAPI, blue) in IPEC-J2 cells. Scale bar = 50 μm. Data were expressed as mean ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: Infection, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Translocation Assay

Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: Quercetin alleviates TGEV-induced inflammatory responses by improving mitochondrial function. IPEC-J2 cells were pretreated with quercetin (80 μM) for 1 h and subsequently infected with TGEV (MOI = 1) for 24 h. To modulate mitochondrial function, cells were co-treated with either NAC (5 mM) or rotenone (1 μM). (A) Flow cytometric analysis of mitochondrial parameters following NAC treatment (n = 3). (B) Western blot analysis of phosphorylated IκBα, NF-κB p65, and NLRP3 in cells co-treated with quercetin and NAC. (C) Flow cytometric analysis of mitochondrial parameters following rotenone treatment (n = 3). (D) Western blot analysis of inflammatory signaling markers in cells co-treated with quercetin and rotenone. (E) Comparative flow cytometric assessment of MMP, mitochondrial biomass, and mtROS in cells treated with quercetin alone or in combination with NAC or rotenone (n = 3). (F) Immunofluorescence staining of NF-κB p65 (green) and nuclei (DAPI, blue), showing subcellular localization across different treatment groups. Scale bar = 5 μm. Data were expressed as mean ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: Infection, Western Blot, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: Quercetin mitigates TGEV-induced inflammation by suppressing excessive PINK1/Parkin-dependent mitophagy. IPEC-J2 cells were pretreated with quercetin (80 μM) for 1 h before infection with TGEV (MOI = 1) for 24 h. To modulate mitophagy, cells were treated with CCCP (5 μM) or Mdivi-1 (10 μM) as indicated. For autophagic flux detection, cells were infected with mCherry-GFP-LC3 adenovirus (MOI = 10) for 24 h prior to quercetin or virus exposure. (A) Western blot analysis of p62, Beclin1, LC3, PINK1, and Parkin at 0, 6, 12, and 24 h after TGEV infection. (B) Confocal imaging of IPEC-J2 cells infected with mCherry-GFP-LC3 adenovirus showing autophagosome (yellow) and autolysosome (red) signals following TGEV infection. Scale bar = 5 μm. (C) MitoTracker Red and LysoTracker Green co-staining with DAPI counterstaining in TGEV-infected cells to visualize mitochondrial-lysosome colocalization. Scale bar = 5 μm. (D) Western blot analysis of p62, LC3, PINK1, and Parkin in TGEV-infected cells treated with quercetin. (E) Confocal images of mCherry-GFP-LC3-expressing IPEC-J2 cells following quercetin treatment in TGEV-infected cells. Scale bar = 5 μm. (F) MitoTracker and LysoTracker co-staining in TGEV-infected cells treated with quercetin. Scale bar = 5 μm. (G) Flow cytometric analysis of MMP, mtROS, and mitochondrial biomass in TGEV-infected cells treated with quercetin, CCCP, or Mdivi-1 (n = 3). (H) Western blot analysis of phosphorylated p65, phosphorylated IκBα, and NLRP3 under different mitophagy modulation conditions. (I) Immunofluorescence staining of NF-κB p65 (green) and DAPI (blue) to assess nuclear translocation status across treatment groups. Scale bar = 5 μm. Data were expressed as mean ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: Infection, Virus, Western Blot, Imaging, Staining, Expressing, Immunofluorescence, Translocation Assay

Journal: International Journal of Biological Sciences

Article Title: Quercetin Protects Against Transmissible Gastroenteritis Virus-Induced Intestinal Inflammation by Modulating Mitophagy-Driven Mitochondrial Dysfunction

doi: 10.7150/ijbs.116855

Figure Lengend Snippet: Quercetin protects against TGEV-induced intestinal inflammation by suppressing mitophagy-driven mitochondrial dysfunction in vivo . (A) Experimental design schematic illustrating the piglet model with dietary quercetin supplementation and TGEV oral challenge. (B) Diarrhea incidence rate of piglets from control, TGEV-infected, and TGEV+Quercetin groups. (C) mRNA expression of tight junction proteins ZO-1 and Claudin-1 in jejunal mucosa. (D) mRNA expression of diarrhea-associated electrolyte and nutrient transporters SLC9A3 ( Nhe3 ) and SLC5A1 ( Sglt1 ) in jejunal mucosa. (E) Flow cytometric analysis of TGEV infection in jejunal epithelial cells using an anti-TGEV nucleocapsid antibody labeled with Alexa Fluor 488. (F) Flow cytometric quantification of DAPI-positive dead cells and p-P65-positive inflammatory epithelial cells in jejunal epithelial cells. (G) mRNA expression levels of pro-inflammatory cytokines ( IL-1β , IL-6 , TNF-α , and NLRP3 ) visualized as a heatmap. (H) Flow cytometric analysis of mitochondrial function markers, including mtROS, MMP, and mtBiomass. (I) Western blot analysis and quantitative densitometry of proteins involved in mitophagy (PINK1, Parkin, LC3-II/I) and inflammation (P-P65, P-IκBα, NLRP3). Data were expressed as mean ±SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After washing, cells were treated with fluorescent secondary antibodies for 1 h in the dark, followed by DAPI staining (Beyotime, Shanghai, China; P0131) for 10 min.

Techniques: In Vivo, Control, Infection, Expressing, Labeling, Western Blot

Journal: Nucleus

Article Title: Discovery of novel murine PML isoforms

doi: 10.1080/19491034.2026.2646815

Figure Lengend Snippet: Formation of PML NBs for individual mPML isoforms. Confocal microscopy visualization of individual mPML isoforms expressed as fusion proteins with C-terminal tRFP tag (cyan) in Pml +/+ or Pml −/− MEFs. Endogenous mPML proteins (yellow) and SUMO-1 proteins (magenta) were immunostained using specific antibodies. Cell nuclei were visualized by DAPI staining (borders of the nuclei indicated by a dashed line in the merge). Importantly, the anti-mPML antibody also detects the transiently expressed mPML-tRFP isoforms. Scale bar, 5 μm.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal antibody against mouse PML protein (Merck, Cat. No. MAB3738), rabbit monoclonal antibody against mouse SUMO-1 protein (ABclonal, Cat. No. A19121) and rabbit polyclonal antibody against mouse GAPDH protein (Merck, Cat. No. G9545).

Techniques: Confocal Microscopy, Staining

Journal: Nucleus

Article Title: Discovery of novel murine PML isoforms

doi: 10.1080/19491034.2026.2646815

Figure Lengend Snippet: The effect of arsenic treatment on mPML NB dynamics. Pml −/− MEFs transiently expressing individual mPML isoforms or control Pml +/+ MEFs or 3T3 cells were mock-treated or treated with 5 µM ATO for 1 h. Subsequently, the cells were fixed and stained using specific antibodies against mPML (green) and SUMO-1 (magenta) proteins. Cell nuclei were visualized by DAPI staining (blue). Representative fluorescence microscopy images are shown. Scale bar, 10 μm.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal antibody against mouse PML protein (Merck, Cat. No. MAB3738), rabbit monoclonal antibody against mouse SUMO-1 protein (ABclonal, Cat. No. A19121) and rabbit polyclonal antibody against mouse GAPDH protein (Merck, Cat. No. G9545).

Techniques: Expressing, Control, Staining, Fluorescence, Microscopy