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 of experimental design: EVs isolated from POCD and non-POCD patient(Control) plasma were applied to primary hippocampal neuron or injected into aged mice (five injections, every other day). Behavioral and electrophysiological assessments were conducted from day 11 onward. B , C Characterization of EVs by TEM, NTA, and immunoblotting. D Immunofluorescence analysis of hippocampal tissue showing elevated ACLY and Iba1 expression following EVs POCD administration. n = 5 mice in each group, ** p < 0.01, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. E Co-localization of EAAT1 and p65 in EVs-treated mice. n = 5 mice in each group, * p < 0.05, ** p < 0.01, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. F Glutamate levels were assessed in hippocampal tissue after EVs POCD treatment. * p < 0.05, n = 5 mice in each group/time point, all values represent mean ± standard deviation; two-way ANOVA with factors time and treatment with Newman–Keuls multiple comparison test. G , H Morris water maze performance showing impaired spatial learning and memory after EVs Con /EVs POCD treatment. n = 15 mice in each group, * p < 0.05, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. I Fluoro-Jade B staining revealed increased degenerating neurons in EVs POCD -treated hippocampi. n = 5 mice in each group, * p < 0.05, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. J Electrophysiology showing attenuated fEPSPs and impaired LTP induction in EVs POCD -treated slices. n = 10 mice in each group, ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests.

Article Snippet: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

Techniques: Isolation, Control, Clinical Proteomics, Injection, Western Blot, Immunofluorescence, Expressing, Standard Deviation, Two Tailed Test, Comparison, Staining

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 Representative immunofluorescence images showing staining for ACLY (yellow), microglial marker IBA1 (red), and nuclei marker DAPI (blue) in 20-month-old POCD mice treated with or without PLX5622. Right panels: fluorescence intensity profiles for control and PLX5622-treated groups. Scale bar: 20 μm. n = 5 mice in each group, ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. B Immunofluorescence staining depicting astrocytic marker GFAP (green), glutamate transporter EAAT1 (red), acetylated p65 (yellow), and nuclei marker DAPI (blue). Right panels: corresponding fluorescence intensity profiles comparing control and PLX5622-treated groups. Scale bar: 20 μm. n = 5 mice in each group, * p < 0.05, ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. C Representative immunofluorescence images showing dendritic marker MAP2 (green), microglial marker IBA1 (red), and nuclei marker DAPI (blue). Intensity profiles are presented for both control and PLX5622-treated groups. Scale bar: 50 μm. n = 5 mice in each group, * p < 0.05, ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. D Time course showing glutamate release (% baseline) in hippocampal slices from POCD mice treated with or without PLX5622. Arrows indicate stimulation. n = 5 mice in each group/time point, ** p < 0.01, two-way ANOVA with Newman–Keuls multiple comparison test. E Representative swimming trajectories in Morris water maze tests for POCD mice treated with or without PLX5622. F Quantification of escape latency (left panel) and number of crossings over the platform location (right panel) in the Morris water maze test. n = 15 mice in each group, * p < 0.05, ** p < 0.01; all values represent mean ± standard deviation. independent two-tailed Student’s t tests. G Representative field excitatory postsynaptic potentials (fEPSP) recorded from hippocampal slices (left inset indicates recording and stimulating electrode positions). Traces from control and PLX5622-treated POCD miceshown. “1” = baseline fEPSP trace before HFS (pre-LTP induction); “2” = fEPSP trace recorded at the late phase after HFS. H Quantification of normalized fEPSP slopes (left panel) and long-term potentiation (LTP) time course following high-frequency stimulation (HFS) (right panel). n = 10 mice in each group, ** p < 0.01; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. Golgi staining illustrating hippocampal dendritic spine morphology ( J ) and representative high-magnification images of dendritic spines ( K ). Scale bars are indicated. Quantitative analysis of spine density ( L ) and mushroom spine density ( M ) per 10 μm dendritic segment. n = 30 dendritic segments analyzed from 5 mice per group, ** p < 0.01; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests.

Article Snippet: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Control, Standard Deviation, Two Tailed Test, Comparison

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: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

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: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

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: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

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: 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 Immunofluorescence staining of ACLY (yellow), microglial marker IBA1 (red), and nuclei (DAPI, blue) in brain tissues from POCD mice treated with EVs Con or EVs M1 (5 µg in 2 μl PBS). Fluorescence intensity profiles presented for each group. Scale bar:20 μm. n = 5 mice per group. * p < 0.05, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. B Immunofluorescence images of astrocytic marker GFAP (green), glutamate transporter EAAT1 (red), acetylated p65 (yellow), and nuclei (DAPI, blue). Corresponding fluorescence intensity profiles are shown for each condition. Scale bar: 20 μm. n = 5 mice per group. * p < 0.05, ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. C Immunofluorescence staining of dendritic marker MAP2 (green), microglial marker IBA1 (red), and nuclei (DAPI, blue). Intensity profiles indicate distribution patterns. Scale bar: 50 μm. n = 5 mice per group. * p < 0.05, *** p < 0.001, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. D Fluoro-Jade B (FJB) staining of hippocampal regions to identify neuronal damage in POCD mice treated with EVs Con or EVs M1 . Quantification of FJB-positive cells shown on the right. Scale bar: 100 μm. n = 5mice per group. ** p < 0.01, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. E Morris water maze test assessing cognitive function in POCD mice treated with EVs Con or EVs M1 . Representative swim paths, escape latency, and platform crossing times presented. n = 15mice in each group, * p < 0.05, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. Golgi staining images illustrating hippocampal dendritic spine density ( F ) and high-magnification views of dendritic spines ( G ). Quantitative analysis of spine number and mushroom spine number per 10 μm dendritic segment shown. Scale bar indicated. n = 30 dendritic segments analyzed from 5 mice per group. * p < 0.05, *** p < 0.0001, all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. Transmission electron microscopy (TEM) images of synaptic ultrastructure, highlighting synaptic morphology and postsynaptic density (PSD) thickness ( H ), and quantification of synapse number per field ( I ). Scale bars: 500 nm. n = 5 mice in each group, * p < 0.05, all values represent mean ± standard deviation. independent two-tailed Student’s t tests.

Article Snippet: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Standard Deviation, Two Tailed Test, Transmission Assay, Electron Microscopy

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 verifying ACLY knockout in EVs derived from microglia. EV markers CD63 and CD81, and negative marker GM130 are included. B Representative TEM images illustrating the morphology of EVs M1-ACLY siRNA NC and EVs M1-ACLY KO . Scale bar: 100 nm. C Nanoparticle tracking analysis showing particle size distribution profiles of EVs M1-ACLY siRNA NC and EVs M1-ACLY KO . D Immunofluorescence staining for GFAP (astrocyte marker, green), EAAT1 (red), acetylated p65 (yellow), and DAPI (nuclei, blue) in brain tissues from POCD mice treated with EVs M1-ACLY siRNA NC or EVs M1-ACLY KO (5 µg in 2 μl PBS). Corresponding intensity profiles are presented. Scale bar: 20 μm. n = 5 mice per group. * p < 0.05, ** p < 0.01, all values represent mean ± standard deviation. independent two-tailed Student’s t tests. E Fluoro-Jade B (FJB) staining assessing neuronal damage in the hippocampus of POCD mice receiving EVs M1-ACLY siRNA NC or EVs M1-ACLY KO treatment. Quantitative analysis presented. Scale bar, indicated. ** p < 0.05, n = 5 mice per group; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. F Morris water maze test depicting cognitive performance through representative swim paths, number of crossings over the previous platform location, and escape latency in POCD mice treated with EVs M1-ACLY siRNA NC or EVs M1-ACLY KO . * p < 0.05, n = 15 mice in each group; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. G Time course of glutamate release (% baseline) measured in hippocampal slices following stimulation in POCD mice treated with EVs M1-ACLY siRNA NC or EVs M1-ACLY KO . * p < 0.05, ** p < 0.01, n = 5 mice in each group/time point, all values represent mean ± standard deviation; two-way ANOVA with Newman–Keuls multiple comparison test. H , I Representative field excitatory postsynaptic potentials (fEPSP) recorded from hippocampal slices and quantification of normalized fEPSP slopes in POCD mice treated with EVs M1-ACLY siRNA NC or EVs M1-ACLY KO . ** p < 0.01, n = 10; all values represent mean ± standard deviation. Independent two-tailed Student’s t tests. “1” = baseline fEPSP trace before HFS (pre-LTP induction); “2” = fEPSP trace recorded at the late phase after HFS.

Article Snippet: Transfection complexes were prepared by mixing 2 μg of either p65 -targeting shRNA(Genechem, Shanghai, China) or non-targeting control shRNA with 5 μL of Lipofectamine 3000 reagent (#L3000015, Invitrogen, USA) in 250 μL Opti-MEM medium.

Techniques: Western Blot, Knock-Out, Derivative Assay, Marker, Immunofluorescence, Staining, Standard Deviation, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: LCK deficiency in CD8 T cells leads to reduced proliferation and increased effector T-cell formation in mice

doi: 10.1101/2025.08.28.672860

Figure Lengend Snippet: (A-E) Cells isolated from spleens and lymph nodes of Ly5.1 WT OT-I Rag2 -/- and Ly5.2 Lck -/- OT-I Rag2 -/- mice were mixed at 1:9 (B-C) or 2:8 (D-E) ratio and co-cultured with OVA peptide-loaded T2-Kb cells at 2:1 ratio (B-C) or activated with 1 µM OVA peptide (D-E) for 1 hour. (A) A schematic illustration of the experiments with T2-Kb cells. (B-C) Signaling intermediates were quantified by flow cytometry. Representative histograms (B) and aggregate results of the percentage of positive cells (C) are shown. n = 5 independent experiments for IKBa or 3 for other signaling intermediates. (D-E) OT-I T cells were activated or not and stained for CD8, TCRβ, DAPI and NFAT or NFκB. Nuclear translocations of NFAT and NFκB were analyzed by imaging flow cytometry (for gating strategy see Fig. S4C). Representative images (D) and the aggregate data of the nuclear translocation scores (E) are shown. Scale bar 7 µm. n = 3 independent experiments. Mean±SEM. Statistical significance was determined using two-tailed paired t test.

Article Snippet: The following antibodies were purchased from Cell signaling: Phospho-p44/42 MAPK XP (Cat# 4370, IgG), Phospho-c-Jun (Cat# 3270, IgG), NFAT1 XP (Cat# 5861S, IgG), IκBα in AF 647 (Cat# 8993, IgG1), Phospho-S6 Ribosomal Protein (Ser235/236) (Cat# 4858, IgG) or Santa Cruz: NFκB p65 (Cat# sc-372, clone C-20, IgG).

Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, Imaging, Translocation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Chlamydia iron starvation links nutritional immunity to pathogen recognition

doi: 10.1101/2025.08.08.669282

Figure Lengend Snippet: A) p65 mRNA expression in HeLa cells at 48h post-transfection, comparing mock and infected cells, with or without bipyridyl treatment (n=3). B) Western blot analysis of intracellular p65 protein levels in HeLa cells at 48h post-transfection using β- actin as loading control (n=2). C) TNF transcript levels in HeLa cells at 48h post-transfection comparing mock and CtrL2 -infected cells under untreated or iron-starved conditions, as measured by qPCR (n=3). D) Intracellular TNF expression in HeLa cells with p65 knockdown at 48h post-transfection detected by western blotting, using vinculin as the loading control and chlamydial MOMP as the infection control (n=1). E) TNF protein levels in HeLa cell supernatants at 48h post-transfection measured using Multiplexing LASER Bead Technology in mock-or CtrL2 -infected with p65 knockdown, with or without bipyridyl treatment (100µM), (n=3). F) Micrographs representing NFκB (red) translocation to the nucleus (blue) in uninfected and CtrL2 infected (green) HeLa cells. G) Quantification of NFκB nuclear translocation in (F) expressed as the ratio of nuclear to cytosolic NFκB fluorescence. 50 random cells were measured per treatment group. H) TNF transcript levels in GSK717-pretreated End1 cells analyzed by qPCR at 24hpi under infected and iron-starved conditions (n=3). I) TNF levels in GSK717 pre-treated End1 cell supernatants at 24hpi, measured using Multiplexing LASER Bead Technology in mock-or CtrL2 -infected cells, with or without bipyridyl treatment (n=3). J) Quantification of NFκB nuclear translocation in End1 cells expressed as nuclear to cytoplasmic ratios for the micrographs shown in K) when GSK717-pretreated End1 cells were infected under untreated and iron-starved conditions. 50 random cells were measured per treatment group. Statistical significance determined by two-way ANOVA and Tukey’s multiple comparisons test. ****p<0.0001; ***p<0.0002; **p<0.002; *p<0.03; ns, not significant.

Article Snippet: HeLa cells at approximately 50% confluency were transfected with SignalSilence Control siRNA (Cell Signaling Technology 6201S) or SignalSilence NF-κB p65 siRNA (Cell Signaling Technology 6261S) using RNAiMAX (Thermo Fisher Scientific, 13778030), following the manufacturer’s protocol, at an optimized concentration of 10nM.

Techniques: Expressing, Transfection, Infection, Western Blot, Control, Knockdown, Multiplexing, Translocation Assay, Fluorescence