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