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: Molecular Cancer

Article Title: Integrated single-cell and spatial transcriptomic profiling decodes lineage plasticity and immune microenvironment remodeling in prostate cancer progression

doi: 10.1186/s12943-026-02617-6

Figure Lengend Snippet: Functional characterization of FOSL1 as a key regulator in double-negative prostate cancer subtype. A Transcription factor activity in malignant epithelial subtypes. Heatmap displaying the top transcription factors with the highest inferred activity (based on SCENIC analysis) in each of the four malignant epithelial subtypes. Color scale (blue to red) represents low to high regulon activity. B Spatial activity of FOSL1 across subtypes. UMAP of malignant epithelial cells colored by the SCENIC-derived regulon activity score of FOSL1, showing its specific enrichment in Subtype 4. Gradient from white to blue indicates low to high activity. C Effect of FOSL1 knockdown on cell proliferation (CCK-8 assay). Growth curves of PC-3 cells transfected with control siRNA or three independent siRNAs targeting FOSL1. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.01 (two-way ANOVA). D Effect of FOSL1 knockdown on clonogenic survival. Representative images of colony formation assays for PC-3 cells treated as in (C). E Effect of FOSL1 knockdown on apoptosis. (Left) Representative flow cytometry plots of Annexin V/PI staining. Quadrants: viable cells (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺). (Right) Quantification of total apoptotic cells (early + late apoptosis). Data are mean ± SD; *** P < 0.001, ** P < 0.01 (one-way ANOVA). F Identification of FOSL1-regulated DNPC genes. Venn diagram showing the overlap between predicted FOSL1 target genes (from SCENIC) and the DNPC gene signature, yielding 8 candidate genes. G FOSL1 binding to candidate gene promoters (ChIP-qPCR). ChIP-qPCR analysis showing enrichment of FOSL1 at the promoters of the 8 candidate genes in control vs. FOSL1-knockdown PC-3 cells. Results are presented as % input. Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). H Expression of candidate genes upon FOSL1 knockdown (qRT-PCR). mRNA expression levels of four selected candidate genes in PC-3 cells after FOSL1 knockdown. Data are normalized to control and presented as mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). I Correlation between FOSL1 and HMGA1 expression. Scatter plot showing a significant positive correlation between FOSL1 and HMGA1 expression across all malignant epithelial cells (n = 152,872). The red line indicates the linear regression fit (R² = 0.212, P < 0.0001, Pearson correlation). J Spatial co-expression of FOSL1 and HMGA1. (Left three panels) Spatial mapping of FOSL1 and HMGA1 expression and their co-localization in a representative section (Patient 01). Red spots indicate high co-expression, yellow indicates high FOSL1 alone, green indicates high HMGA1 alone, and gray indicates low expression of both. (Right panel) Correlation analysis of FOSL1 and HMGA1 expression specifically within the co-expressing spots (red spots, left panel), showing a strong positive correlation (R = 0.923, P = 1.28e-10, Pearson correlation). K - M Western blot analysis of downstream pathways. Protein levels of (K) stemness markers (CD44, OCT4, SOX2, NANOG), (L) EMT markers (ZO-1, N-cadherin, E-cadherin, Vimentin, SNAIL), and (M) DNPC markers (DSG3, KRT6A, KRT5), along with FOSL1 and HMGA1, in PC-3 cells under four conditions: Control, FOSL1 knockdown (siFOSL1), HMGA1 overexpression (oeHMGA1), and FOSL1 knockdown combined with HMGA1 rescue (siFOSL1 + oeHMGA1). GAPDH served as a loading control. N Rescue of proliferation by HMGA1 overexpression. CCK-8 proliferation assay of PC-3 cells under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-way ANOVA). O Rescue of apoptosis by HMGA1 overexpression. Quantification of total apoptosis (Annexin V⁺ cells) by flow cytometry under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (one-way ANOVA). P Pharmacological inhibition of FOSL1 reduces HMGA1. Western blot showing dose-dependent decrease of FOSL1 and its downstream target HMGA1 in PC-3 cells treated with increasing concentrations (0, 0.2, 2, 5, 10 µM) of the FOSL1 degrader T-5224. Q In vivo combination therapy schematic. Workflow of the xenograft study. Nude mice bearing PC-3 subcutaneous tumors were treated starting at day 13 post-inoculation with vehicle, Docetaxel (1 mg/kg), FOSL1 degrader T-5224 (10 mg/kg), or the combination via intraperitoneal injection every 48 h. Treatment continued until day 28. R Representative images of resected tumors. Photographs of excised tumors from each treatment group at the study endpoint. S Tumor growth curves. Tumor volume (mm³) was measured over time for each treatment group. Data are presented as mean ± SEM; *** P < 0.001 (two-way ANOVA). T Tumor weight at endpoint. Final tumor weights (g) for each group. Data are mean ± SEM; *** P < 0.001, ** P < 0.01 (one-way ANOVA). U Assessment of tumor cell proliferation (IHC). Representative immunohistochemistry images showing Ki67 expression in tumor sections from each treatment group. Scale bar, 100 μm

Article Snippet: HMGA1 overexpression plasmid (Cat: HG11613-NF), and the empty vector plasmid pCMV3-N-FLAG were purchased from SinoBiological.

Techniques: Functional Assay, Activity Assay, Derivative Assay, Knockdown, CCK-8 Assay, Transfection, Control, Flow Cytometry, Staining, Binding Assay, ChIP-qPCR, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Proliferation Assay, Inhibition, In Vivo, Injection, Immunohistochemistry