Journal: Brain

Article Title: Occludin modulates HIV and ischaemic stroke response via the mitochondrial antiviral signalling pathway

doi: 10.1093/brain/awaf262

Figure Lengend Snippet: Occludin regulates HIV-1 infection through the ISG antiviral gene expression. Pericytes were transfected with Ocln -siRNA, ZO-1 siRNA or PCMV3-OCLN vectors, and the expression of ISG15 ( A ), IFIT1 ( C ), MX1 ( E ), MX2 ( F ), USP18 ( G ) and HERC5 ( H ) was evaluated by quantitative PCR (qPCR) and western blotting. Pericytes were transfected with ISG15 -siRNA, MX1 -siRNA, MX2 -siRNA and Ocln -siRNA. Next, cells were either mock-infected or infected with 60 ng/ml HIV-1 p24 for 12 h. p24 levels were analysed in cell culture media by ELISA as a marker of HIV infection ( I ). n = 3–8 per group. RNA was extracted from isolated microvessels of age- and sex-matched Ocln −/− , Ocln +/− and Ocln +/+ mice ( n = 6–8 animals per group). ISG15 ( B ), IFIT1 ( D ), IFNα5 ( J ), IFNβ1 ( K ) and IFNɣ ( L ) mRNA levels were analysed by qPCR. Graphs indicate the mean ± standard deviation from three independent experiments. Individual animal data-points are denoted by blue ( Ocln +/+ ), green ( Ocln +/− ) and red ( Ocln −/− ) filled circles. **** P < 0.0001, *** P = 0.0002, ** P = 0.003, * P < 0.0449; one-way ANOVA. ISGs = interferon-stimulated genes.

Article Snippet: For Ocln overexpression, cells were transfected with the PCMV3-OCLN plasmid (Sino Biological, Cat. No. HG15134-UT) or PCMV3 (Sino Biological, Cat. No. CV011) as a negative control.

Techniques: Infection, Gene Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Marker, Isolation, Standard Deviation

Journal: Brain

Article Title: Occludin modulates HIV and ischaemic stroke response via the mitochondrial antiviral signalling pathway

doi: 10.1093/brain/awaf262

Figure Lengend Snippet: Occludin regulates the RIG-1-like receptor pathway signalling. ( A ) A diagram of the antiviral RIG-1 pathway. Activated RIG-I and MDA5 molecules translocate to the mitochondria and interact with the MAVS adaptor. MAVS activates the downstream molecules TBK1/IKKε kinases, IRF3 and IRF7, inducing the transcriptional expression of IFN-α/β. Created in BioRender. Torices, S. (2025) https://BioRender.com/votsmme . Pericytes were transfected with Ocln -siRNA, ZO-1 siRNA or PCMV3-OCLN vectors as in . The expression of RIG-1 ( B ), IFIH1 ( D ), MAVS ( F ), TRAF3 ( H ), IRF3 and P -IRF3 ( J ), as well as TBK1 and p-TBK1 ( K ), was evaluated by qPCR and western blotting. ( L ) Pericytes were transfected with RIG-1 -siRNA, and p24 levels were assessed as in . ( M and N ) Pericytes were transfected with the PCMV3-OCLN vector and HIV-1 or mock-infected as in . IRF3 and IRF7 mRNA levels were measured by qPCR. n = 6−8 per group. ( C , E , G and I ) RNA was extracted from isolated microvessels of age- and sex-matched Ocln −/− , Ocln +/− and Ocln +/+ mice ( n = 5–11 animals per group) as in . RIG-1 ( C ), IFIH1 ( E ), MAVS ( G ) and TRAF3 ( I ) mRNA expression levels were measured by qPCR. Graphs indicate the mean ± standard deviation from three independent experiments. Individual animal data-points are marked by blue ( Ocln +/+ ), green ( Ocln +/− ) and red ( Ocln −/− ) filled circles. **** P < 0.0001, *** P = 0.0002, ** P = 0.003, * P < 0.0449; one-way ANOVA ( B – L ) and two-way ANOVA ( M and N ).

Article Snippet: For Ocln overexpression, cells were transfected with the PCMV3-OCLN plasmid (Sino Biological, Cat. No. HG15134-UT) or PCMV3 (Sino Biological, Cat. No. CV011) as a negative control.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Infection, Isolation, Standard Deviation

Journal: Brain

Article Title: Occludin modulates HIV and ischaemic stroke response via the mitochondrial antiviral signalling pathway

doi: 10.1093/brain/awaf262

Figure Lengend Snippet: Impact of occludin on mitochondrial dysregulation. Pericytes were transfected with the PCMV3-OCLN vector and HIV-1 or mock-infected as in . FIS1 ( A ), MFF ( C ), MFN2 ( E ) and OPA1 ( G ) mRNA and protein levels were measured by qPCR and western blotting. n = 4–8 per group. In addition, age- and sex-matched Ocln −/− , Ocln +/− and Ocln +/+ mice ( n = 4–11 animals per group) were EcoHIV or mock-infected and RNA was extracted from isolated microvessels. FIS1 ( B ), MFF ( D ), MFN2 ( F ) and OPA1 ( H ) mRNA expression levels were measured by qPCR. Graphs indicate the mean ± standard deviation from three independent experiments. Individual animal data-points are marked by blue ( Ocln +/+ ), green ( Ocln +/− ) and red ( Ocln −/− ) dots. **** P < 0.0001, *** P = 0.0002, ** P = 0.003, * P < 0.0449; one-way ANOVA or two-way ANOVA.

Article Snippet: For Ocln overexpression, cells were transfected with the PCMV3-OCLN plasmid (Sino Biological, Cat. No. HG15134-UT) or PCMV3 (Sino Biological, Cat. No. CV011) as a negative control.

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Isolation, Expressing, Standard Deviation

Journal: Brain

Article Title: Occludin modulates HIV and ischaemic stroke response via the mitochondrial antiviral signalling pathway

doi: 10.1093/brain/awaf262

Figure Lengend Snippet: Impact of occludin on mitochondrial respiratory function. ( A ) Seahorse Cell Mito Stress Test analysis of OCR after transfecting pericytes with different concentrations, 1 µg (+) or 1.5 µg (++) of PCMV3-OCLN vector per 10 6 cells. ( B ) Quantitative measurements of basal respiration, non-mitochondrial oxygen consumption, maximal respiration, ATP production, proton leak and spare respiration capacity were performed. ( C ) Confocal microscopy images of pericytes after OCLN overexpression. Cells were stained with DAPI (blue), OCLN (red) and TOM20 (green) to track the mitochondrial membrane. ( D and E ) Mitochondria were skeletonized, and mitochondrial mean branch ( D ) and footprint ( E ) were quantified using the MiNA plug-in for ImageJ. ( F ) Measurement of mitochondrial superoxide was determined with MitoSOX Red after transfecting pericytes with different concentrations of PCMV3-OCLN vector. The fluorescence intensity of MitoSOX Red was normalized to the protein levels. ( G ) Total TOM20 protein levels were measured by western blotting. Graphs indicate the mean ± standard deviation from three independent experiments. *** P = 0.0002, ** P = 0.003, * P < 0.0449, n = 4–8 per group; one-way ANOVA. Scale bars = 30 µm. DAPI = 4′,6-diamidino-2-phenylindole; MiNA = mitochondrial network analysis; OCR = oxygen consumption rate.

Article Snippet: For Ocln overexpression, cells were transfected with the PCMV3-OCLN plasmid (Sino Biological, Cat. No. HG15134-UT) or PCMV3 (Sino Biological, Cat. No. CV011) as a negative control.

Techniques: Plasmid Preparation, Confocal Microscopy, Over Expression, Staining, Membrane, Fluorescence, Western Blot, Standard Deviation

Journal: Brain

Article Title: Occludin modulates HIV and ischaemic stroke response via the mitochondrial antiviral signalling pathway

doi: 10.1093/brain/awaf262

Figure Lengend Snippet: Impact of occludin on autophagy and apoptosis regulation. Pericytes were transfected with the PCMV3-OCLN vector as in . LC3B-I and LC3B-II ( A ), p62 ( B ), NDP52 ( C ), PGC1α ( D ), BAX ( E ) and BCL2 ( G ) mRNA and protein levels were measured by qPCR and western blotting. n = 4–8 per group. BAX ( F ) and BCL2 ( H ) mRNA expression levels from extracted microvessels isolated from age- and sex-matched Ocln −/− , Ocln +/− and Ocln +/+ mice ( n = 7–10 animals per group). Graphs indicate the mean ± standard deviation from three independent experiments. Individual animal data-points are denoted by blue ( Ocln +/+ ), green ( Ocln +/− ) and red ( Ocln −/− ) filled circles. **** P < 0.0001, *** P = 0.0002, ** P = 0.003, * P < 0.0449; one-way ANOVA.

Article Snippet: For Ocln overexpression, cells were transfected with the PCMV3-OCLN plasmid (Sino Biological, Cat. No. HG15134-UT) or PCMV3 (Sino Biological, Cat. No. CV011) as a negative control.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Isolation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Spermidine-Eugenol Supplement Preserved Inflammation-Challenged Intestinal Cells by Stimulating Autophagy

doi: 10.3390/ijms24044131

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) staining, and LC3-II, occludin, and mucus expression of 3D intestinal equivalents (Caco-2/U937/L929 co-cultures) exposed to a 24 h pretreatment of either 1.2 spermidine (SPD) and 0.2 mM eugenol (EUG) or supplement (1.2 µM SPD + 92 µM EUG) prior to the addition of lipopolysaccharide (LPS, 1 ng/mL) for a further 24 h compared to the untreated control (CTRL) and LPS treatments alone. The magnification was ×60. ( B ) Quantification of LC3-II-red chromogen staining, ( C ) occludin–red chromogen staining ( D ) mucus expression from Alcian Blue/Periodic Acid-Schiff Stain (PAS) staining of the Caco-2 cells. Significant differences were represented as follows: ** 0.001 < p < 0.01, **** p < 0.0001. The black dots indicate the positioning of the individual replicates within the bar for each sample.

Article Snippet: LC3-II and occludin antibodies were from Novus Biologicals (Milan, Italy), whereas the SQSTM1/P62 was from ABclonal Technology (Düsseldorf, Germany).

Techniques: Staining, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Indicators of Blood-Brain Barrier Breakdown and Neuronal Injury in Pregnancy Complicated by Fetal Growth Restriction

doi: 10.3390/ijms232213798

Figure Lengend Snippet: Serum concentrations of biochemical parameters in pregnancies complicated by fetal growth restriction (FGR) and physiological pregnancy. The statistically significant differences between groups are highlighted in bold.

Article Snippet: Recombinant human OCLN (Recombinant Human Occludin GST (N-Term) Protein, Novus Biologicals, Littleton, CO, USA) and recombinant human CLN5 (Recombinant Human Claudin-5 GST (N-Term) Protein, Novus Biologicals, Littleton, CO, USA) served as standards.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Molecular Indicators of Blood-Brain Barrier Breakdown and Neuronal Injury in Pregnancy Complicated by Fetal Growth Restriction

doi: 10.3390/ijms232213798

Figure Lengend Snippet: Placental expression of tight junction proteins in pregnancies complicated by fetal growth restriction (FGR) and physiological pregnancy.

Article Snippet: Recombinant human OCLN (Recombinant Human Occludin GST (N-Term) Protein, Novus Biologicals, Littleton, CO, USA) and recombinant human CLN5 (Recombinant Human Claudin-5 GST (N-Term) Protein, Novus Biologicals, Littleton, CO, USA) served as standards.

Techniques: Expressing

Journal: Oncogene

Article Title: Endoplasmic reticulum protein 29 regulates epithelial cell integrity during the mesenchymal-epithelial transition in breast cancer cells.

doi: 10.1038/onc.2012.149

Figure Lengend Snippet: Figure 4. ERp29 overexpression increases the protein expression of tight junction components, ZO-1 and occludin, and induces their accumulation at the cell membrane and at cell–cell contacts. (a) Overexpression of ERp29 in both MDA-MB-231 cells and BT549 cells promotes an increase in the protein expression of ZO-1 and occludin, but not their mRNA levels relative to the empty-vector-transfected Ctrl cells. (b) Immunofluorescence staining showed that overexpression of ERp29 led to the enhanced expression and distribution of ZO-1 at the membrane and the cell–cell contacts in the clones of both cell-types (clones B and E, A and K). Similarly, ERp29 overexpression significantly promoted the expression and localization of occludin at the membrane and the cell–cell contacts, particularly in ERp29-overexpressing BT549 cells (A and K). Arrows indicate the localization of ZO-1 (red) and occludin (green) at the membrane and the cell–cell contacts. DAPI (blue) was used for nuclear staining. Original magnification: 60. Data represent mean±s.d. based on three independent experiments. **Po0.01; ***Po0.001.

Article Snippet: Reagents and antibodies The following primary antibodies were used: ERp29 (Cat. No: NB300-523), Par3 (Cat. No: NB110-55469), occludin (Cat. No: H00004950-M01) and aPKC (Cat. No: H00005584-M01; Novus Biological, Littleton, CO, USA); Scribble (Cat. No: MAB1820) and vimentin (Cat. No: AB1620) (Millipore, Temecula, CA, USA); Snai1 (Cat. No: PAB7104), Slug (Cat. No: H00006591-M07), ZEB1 (Cat. No: PAB11610) and ZEB2 (Cat. No: PAB19269; Abnova, Taipei, Taiwan); E-cadherin (Cat. No: 3195), MLC (Cat. No: 3672), phosphorylated-MLC (pMLC; Cat. No: 3675), ZO-1 (Cat. No: 5406) and b-actin (Cat. No: 4967; Cell Signaling Technology, Beverly, MA, USA); fibronectin (Cat. No: ab18265), CK19 (Cat. No: ab15463) and Twist (Cat. No: ab73163; Abcam Inc., Cambridge, MA, USA).

Techniques: Over Expression, Expressing, Membrane, Plasmid Preparation, Transfection, Staining, Clone Assay