Journal: Frontiers in Genetics

Article Title: Single-cell transcriptomic profiling uncovers key molecular signatures in glioma pathogenesis

doi: 10.3389/fgene.2026.1818742

Figure Lengend Snippet: Differential expression analysis, gene co-expression network, pathway enrichment, and experimental validation. (A) Volcano plot showing differential expression analysis results, with significantly up- and downregulated genes highlighted. (B) Gene co-expression network revealing functional modules centered on hub genes including CLEC12A, ATP13A4, CLU, AQP4, S100A16, and MT3. (C) Three-dimensional PCA visualization showing spatial distribution of 22 cell clusters. (D) Pathway enrichment dot plot identifying significantly enriched glioma-related pathways including cell cycle, Notch signaling, MAPK, and neurogenesis. (E) Cell type expression correlation heatmap showing relationships between astrocytes, pericytes, differentiated cells, progenitor cells, T cells, neural stem cells, and oligodendrocytes. (F) qRT-PCR validation of candidate biomarkers in C8-D1A normal glial cells, GL261 glioma cells, and NP disc cells, showing significant downregulation of Plp1 (0.38-fold) and upregulation of F th1 (2.15-fold) and Gm42418 (5.67-fold) in glioma cells. ***P < 0.001.

Article Snippet: C8-D1A mouse astrocyte cells (ATCC CRL-2541), representing normal glial cells, and GL261 mouse glioma cells (ATCC CRL-2361) were obtained from the American Type Culture Collection.

Techniques: Quantitative Proteomics, Expressing, Biomarker Discovery, Functional Assay, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Astrocytic EAAT1 suppression by EV-ACLY underlies glutamate imbalance and cognitive impairment in POCD

doi: 10.1038/s42003-026-09888-1

Figure Lengend Snippet: A Western blot analysis of ACLY, p65, ac-p65, and EAAT1 protein levels in nuclear (left part) and cytoplasmic fractions (right part) of C8-D1A cells treated with pcDNA3.1(+)-Flag-ACLY (ACLY) or ACLY inhibitor (SB) and stimulated with LPS or NF-κB inhibitor (BZ). Histone H3 and GAPDH served as nuclear and cytoplasmic loading controls, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5 independent biological replicates (independent cell cultures); all values represent mean ± standard deviation, one-way ANOVA with Newman–Keuls multiple comparison test. B Bioinformatics analysis identifying putative NF-κB p65 binding motifs within the SLC1A3 promoter region. Motifs 1 and 2 indicate potential binding sites relative to the transcription start site (TSS). C ChIP assay results indicating enhanced binding of acetylated p65 (ac-p65) to motifs 1 and 2 in the SLC1A3 promoter region compared to the negative control (NC). IgA/G serves as a control antibody. n = 4 independent biological replicates, * p < 0.05, values represent mean ± standard deviation. independent two-tailed Student’s t tests. D Quantitative PCR analysis demonstrating increased SLC1A3 mRNA expression in C8-D1A cells transfected with shp65/NF-κB compared to control (shNC). n = 4 independent biological replicates, * p < 0.05, values represent mean ± standard deviation. Independent two-tailed Student’s t tests.

Article Snippet: To block the NF-κB signaling pathway, C8-D1A cells were treated with bortezomib (BZ, #HY-100227, MedChem Express, USA), an NF-κB inhibitor.

Techniques: Western Blot, Standard Deviation, Comparison, Binding Assay, Negative Control, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Transfection

Journal: Communications Biology

Article Title: Astrocytic EAAT1 suppression by EV-ACLY underlies glutamate imbalance and cognitive impairment in POCD

doi: 10.1038/s42003-026-09888-1

Figure Lengend Snippet: A Schematic illustration of experimental design for co-culture experiments. Astrocytic C8-D1A cells were co-cultured with BV2/BV2 M1 (activated with LPS, 2 μg/ml for 48 h)/BV2 M1 + GW4869 for 48 h. The downstream analyses included Western blot, annexin V/PI assay and immunofluorescence staining for p65 and EAAT1. Created in BioRender. Ding, L. (2026) https://BioRender.com/g1hk8n9 . B Western blot analysis comparing ACLY protein level in C8-D1A after co-cultured with BV2, BV2 M1 , or BV2 M1 + GW4869. ** p < 0.01, n = 5 independent biological replicates; all values represent mean ± standard deviation, one-way ANOVA with Newman–Keuls multiple comparison test. C Flow cytometry analysis (annexin V/PI) assessing cell viability and apoptosis in C8-D1A cells after co-cultured with BV2, BV2 M1 or BV2 M1 + GW4869. D Immunofluorescence staining of C8-D1A cells for NF-κB p65 (green), glutamate transporter EAAT1 (red), and nuclei (DAPI, blue) in different groups. Scale bar: 50 μm. E Fluorescence intensity profiles for p65, EAAT1 and nuclear-to-cytoplasmic ratio of p65 in C8-D1A co-cultured with BV2, BV2 M1 , or BV2 M1 + GW4869. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, n = 5; all values represent mean ± standard deviation, one-way ANOVA with Newman–Keuls multiple comparison test.

Article Snippet: To block the NF-κB signaling pathway, C8-D1A cells were treated with bortezomib (BZ, #HY-100227, MedChem Express, USA), an NF-κB inhibitor.

Techniques: Co-Culture Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Standard Deviation, Comparison, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Astrocytic EAAT1 suppression by EV-ACLY underlies glutamate imbalance and cognitive impairment in POCD

doi: 10.1038/s42003-026-09888-1

Figure Lengend Snippet: A Schematic illustration depicting the experimental approach used to isolate and characterize EVs from LPS-stimulated BV2 microglial cells (EVs M1 ). EVs were analyzed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot, followed by uptake assays and immunofluorescence staining for ac-p65 and EAAT1. B Representative TEM images showing morphology of EVs from control BV2 (EVs Con ) and activated BV2 (EVs M1 ). Scale bar: 100 nm. C Western blot characterization of EV markers (CD63, CD81), GM130 (negative marker), and ACLY protein in lysates and isolated EVs. D NTA particle size distribution profiles for EVs isolated from control (EVs Con ) and activated microglia (EVs M1 ). E Uptake analysis of PKH67-labeled EVs (green) by C8-D1A cells, co-stained with nuclei marker DAPI (blue). Scale bar, 50 μm. F PBS/EVs Con / EVs M1 were labeled with the lipophilic fluorescent tracer Dir, and then were stereotactically injected into C57BL/6. After 24 h, the EVs distribution in vivo was visualized by an IVIS imaging system. G Immunofluorescence staining showing nuclear localization of acetylated p65 (ac-p65, green), EAAT1 expression (red), and nuclei (DAPI, blue) in C8-D1A cells treated with EVs Con or EVs M1 . Scale bar, 50 μm. H , I Fluorescence intensity profiles of EAAT1 and ac-p65 staining in C8-D1A cells treated with EVs Con and EVs M1 . n = 5, * p < 0.05, ** p < 0.01, Values represent mean ± standard deviation. independent two-tailed Student’s t tests.

Article Snippet: To block the NF-κB signaling pathway, C8-D1A cells were treated with bortezomib (BZ, #HY-100227, MedChem Express, USA), an NF-κB inhibitor.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Staining, Control, Marker, Isolation, Labeling, Injection, In Vivo, Imaging, Expressing, Fluorescence, Standard Deviation, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Rhynchophylline attenuates porcine pseudorabies virus-induced astrocyte injury by modulating oxidative stress, inflammation, and metabolic abnormalities

doi: 10.3389/fphar.2026.1782776

Figure Lengend Snippet: The effects of different concentrations of rhynchophylline (RHY) on the viability of C8-D1A cells were assessed using the cell counting Kit-8 (CCK-8) assay. Data are presented as mean ± standard deviation (SD) (n = 6). The same letters indicate no differences between groups ( P > 0.05), while different letters represent significant differences between groups ( P < 0.05).

Article Snippet: The mouse astrocyte cell line C8-D1A was procured from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in high-glucose DMEM supplemented with 10% FBS and 1% P/S (Gibco, Grand Island, NY, United States) at 37 °C in a 5% CO 2 incubator.

Techniques: Cell Counting, CCK-8 Assay, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Rhynchophylline attenuates porcine pseudorabies virus-induced astrocyte injury by modulating oxidative stress, inflammation, and metabolic abnormalities

doi: 10.3389/fphar.2026.1782776

Figure Lengend Snippet: The effects of RHY on the expression levels of the PRV gE gene in C8-D1A cells were evaluated via real-time quantitative polymerase chain reaction (RT-qPCR) at 12, 24, and 48 h post-infection (hpi). Data are presented as mean ± SD (n = 3). Different lowercase letters (a, b, c) indicate significant differences between groups at the same time point ( P < 0.05); different uppercase letters (A, B, C) indicate significant differences across time points within the same group ( P < 0.05).

Article Snippet: The mouse astrocyte cell line C8-D1A was procured from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in high-glucose DMEM supplemented with 10% FBS and 1% P/S (Gibco, Grand Island, NY, United States) at 37 °C in a 5% CO 2 incubator.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection

Journal: Frontiers in Pharmacology

Article Title: Rhynchophylline attenuates porcine pseudorabies virus-induced astrocyte injury by modulating oxidative stress, inflammation, and metabolic abnormalities

doi: 10.3389/fphar.2026.1782776

Figure Lengend Snippet: The effects of RHY on cytokine mRNA levels in C8-D1A cells were assessed. (A–D) The relative mRNA levels of interleukin (IL)-6 (A) , IL-8 (B) , IL-4 (C) , and IL-10 (D) were detected by RT-qPCR. Data are presented as mean ± SD (n = 3). Different lowercase letters (a, b, c) indicate significant differences between groups at the same time point ( P < 0.05); different uppercase letters (A, B, C) indicate significant differences across time points within the same group ( P < 0.05).

Article Snippet: The mouse astrocyte cell line C8-D1A was procured from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in high-glucose DMEM supplemented with 10% FBS and 1% P/S (Gibco, Grand Island, NY, United States) at 37 °C in a 5% CO 2 incubator.

Techniques: Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Rhynchophylline attenuates porcine pseudorabies virus-induced astrocyte injury by modulating oxidative stress, inflammation, and metabolic abnormalities

doi: 10.3389/fphar.2026.1782776

Figure Lengend Snippet: The effects of RHY on oxidative stress indicators in PRV-infected C8-D1A cells were investigated. (A) Reactive oxygen species (ROS) levels were detected by flow cytometry. (B–F) Xanthine oxidase (XOD) activity (B) , myeloperoxidase (MPO) activity (C) , nitric oxide (NO) content (D) , malondialdehyde (MDA) activity (E) , and superoxide dismutase (SOD) activity (F) were measured using biochemical assay kits. Data are presented as mean ± SD (n = 3). Different lowercase letters (a, b, c) indicate significant differences between groups at the same time point ( P < 0.05); different uppercase letters (A, B, C) indicate significant differences across time points within the same group ( P < 0.05).

Article Snippet: The mouse astrocyte cell line C8-D1A was procured from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in high-glucose DMEM supplemented with 10% FBS and 1% P/S (Gibco, Grand Island, NY, United States) at 37 °C in a 5% CO 2 incubator.

Techniques: Infection, Flow Cytometry, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Rhynchophylline attenuates porcine pseudorabies virus-induced astrocyte injury by modulating oxidative stress, inflammation, and metabolic abnormalities

doi: 10.3389/fphar.2026.1782776

Figure Lengend Snippet: Metabolomic analysis of RHY’s effect on reversing PRV-induced metabolic dysregulation in C8-D1A cells was performed. (A–C) Principal component analysis (PCA) score plots of the control group (Ctrl), PRV-infected group, and RHY-treated group at 12 (A) , 24 (B) , and 48 (C) hours post-infection (hpi). (D–F) Partial Least Squares Discriminant Analysis (PLS-DA) score plots of the Ctrl, PRV, and RHY groups at 12 (D) , 24 (E) , and 48 (F) hours post-infection. (G,H) Volcano plots and heatmaps of differential metabolites between the Ctrl and PRV_12h groups (G) , as well as between PRV_12h and RHY_12h groups (H) . (I,J) Volcano plots and heatmaps of differential metabolites between the Ctrl and PRV_24h groups (I) , as well as between PRV_24h and RHY_24h groups (J) . (K,L) Volcano plots and heatmaps of differential metabolites between the Ctrl and PRV_48h groups (K) , as well as between PRV_48h and RHY_48h groups (L) .

Article Snippet: The mouse astrocyte cell line C8-D1A was procured from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in high-glucose DMEM supplemented with 10% FBS and 1% P/S (Gibco, Grand Island, NY, United States) at 37 °C in a 5% CO 2 incubator.

Techniques: Metabolomic, Control, Infection