Journal: Acta Pharmaceutica Sinica. B

Article Title: Acteoside ameliorates hepatocyte ferroptosis and hepatic ischemia-reperfusion injury via targeting PCBP2

doi: 10.1016/j.apsb.2025.03.002

Figure Lengend Snippet: The hepatocyte specific knockout of PCBP2 interferes the therapeutic effects of ACT. (A) The particle size, (B) PDI, zeta potential and EE (%) of LNPs-si-PCBP2. (C) The stability of LNPs-si-PCBP2 in PBS and cell culture media. (D) The release behaviors of free si-RNA and LNPs-si-PCBP2. (E) Flow chart of ApoE-LNPs animal experiment. (F) The content of Fe 3+ , Fe 2+ , Cys and GSH in mouse livers. Statistical significance: ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with the sham group ( n = 6). Representative images of (G) H & E staining (scale bar = 40 μm), (H) PPB staining (scale bar = 40 μm), (I) immunofluorescence staining for PCBP2/SLC3A2/SLC7A11 (scale bar = 40 μm), (J) immunofluorescence staining for HMGB1 (scale bar = 40 μm) and (K) CD68 (scale bar = 40 μm) in mouse livers. (L) The Relative mRNA levels of slc3a2 , slc7a11 , p300 , hmgb1 and F4/80 were measured by qPCR and further normalized with hprt1 in mouse livers. Statistical significance: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with the sham group ( n = 6).

Article Snippet: The purified apolipoprotein E (ApoE) (MedChemExpress, NJ, USA) was then incubated with iminothiolane hydrochloride (2-IT) at a molar ration 1:2 and dissolved in phosphate-buffered saline at 25 °C for 90 min.

Techniques: Knock-Out, Zeta Potential Analyzer, Cell Culture, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: APOE regulates the transport of GM1

doi: 10.1101/2024.04.02.587789

Figure Lengend Snippet: (a) Schematic illustration of determining the binding affinity between lipid structure and APOE using MST. (b) The binding affinity Kd between APOE3 and lipid structures with different compositions. (n>=4) (c) The binding affinity between APOE3 and lipid structures with different GM1 concentrations. (n>=3) (d) The binding affinity between APOE4 and lipid structures with different compositions. (n>=5) (e) The binding affinity between APOE4 and lipid structures with different GM1 concentrations. (n>=5) P-value: ns (0.05 < p <= 1), * (0.01 < p <= 0.05, ** (0.001 < p <= 0.01, *** (0.0001 < p <= 0.001, **** (p <= 0.0001). (a) is created with BioRender.com.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: APOE regulates the transport of GM1

doi: 10.1101/2024.04.02.587789

Figure Lengend Snippet: (a) The expression of LDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=4). (b) The expression of VLDLR on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=4). (c) The expression of LRP1 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=3). (d) The expression of ApoER2 on differentiated PC-12 cells, U-87 MG, bEnd.3, and HEK-293 cells (n=3). (e) Schematic illustration of determining the changes of APOE secondary structures. (f) The CD results of APOE 3 under the effect of different GM1 content on the lipid structures (n>=3). (g) The CD results of APOE 4 under the effect of different GM1 content on the lipid structures (n=3). (h) Schematic illustration of determining the binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. (i) The binding affinity between APOE3 and lipid structures with different compositions. (n>=4) (j) The binding affinity between APOE4 and lipid structures with different compositions. (n>=3) P-value: ns (0.05 < p <= 1), * (0.01 < p <= 0.05, ** (0.001 < p <= 0.01, *** (0.0001 < p <= 0.001, **** (p <= 0.0001). (e) and (h) are created with BioRender.com.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Expressing, Binding Assay

Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: LILRB4 shows stronger APOE4‐associated Co‐IP signals in the tested contexts and is upregulated in APOE4 and amyloid‐related settings. (A,B) Reciprocal Co‐IP of human APOE4 with Flag‐tagged human LILRB3 (A) or Flag‐tagged human LILRB4 (B) in HEK293T cells. Schematic representation of Co‐IP between LILRB3/LILRB4 and APOE4 protein. Immunoprecipitation with anti‐APOE or anti‐Flag antibodies, followed by immunoblotting for APOE4 and LILRB3/LILRB4. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (C, D) Reciprocal Co‐IP of endogenous APOE and LILRB4 in microglia isolated from 12‐month‐old APOE4 (C) or APOE3 (D) mice 24 h after intraperitoneal LPS injection. Cell lysates were immunoprecipitated with anti‐APOE or anti‐LILRB4 antibodies and immunoblotted as indicated. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (E) Representative Lilrb4a immunostaining in cortical sections from LPS‐treated APOE3 and APOE4 mice (10×, scale bar = 500 µm); (F) Quantification of Lilrb4a‐positive area in the cortex ( n = 4–5); (G) Schematic diagram of the experimental design; (H) Representative confocal images of X‐34 (blue), APOE (red), and IBA1 (green) staining in cortical sections from 5EL and 5ELKO mice; arrows indicate plaque‐associated microglia containing APOE signal (60× oil, scale bar = 50 µm); (I) Quantification of APOE‐positive area within IBA1‐positive microglia surrounding plaques (APOE/IBA1 area) ( n = 9–12); (J, K) Lilrb4a mRNA expression was analyzed by qPCR in cortical tissues from C57 and 5xFAD mice of different ages ( n = 5–8); Unless otherwise specified, experiments not shown as sex‐separated in the figures were performed using male mice only (Applicable to all figure legends). Data are presented as mean ± standard error of the mean (SEM). Each dot represents one mouse. Statistical significance was determined by unpaired two‐tailed Student's t test (F), one‐way ANOVA (J,K), or two‐way ANOVA (I), as appropriate. * p <0.05, ** p <0.01.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Isolation, Injection, Immunostaining, Staining, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: Exploratory bulk RNA‐seq nominates a PPAR‐related signature, and PPAR‑γ activation phenocopies clearance‐associated outcomes. (A) Volcano plot of bulk RNA‐seq data from thalamic tissue of 5EL and 5ELKO mice; colored points indicate genes meeting an exploratory threshold of nominal p <0.05 and absolute fold change >1.2; (B) KEGG pathway enrichment analysis of transcriptomic alterations in 5ELKO versus 5EL mice; (C) qPCR validation of selected genes related to the PPAR signaling pathway, including Apoa1 , Fabp7 , Pck1 , Plin2 , Apoa2 , Pparg , Cyp2e1 in thalamus from 5EL and 5ELKO mice ( n = 4–5); (D) Representative RNAscope images of Pparg mRNA in brain sections from 5EL and 5ELKO mice, combined with X‐34 and IBA1 co‐staining (left) and Imaris‐based reconstruction (right). White arrows indicate PPAR‐γ‐positive signals within microglia (60× oil, scale bar = 20 µm); (E) Quantification of Pparg RNAscope signal volume within plaque‐associated microglia in 5EL and 5ELKO mice; (F) Representative Incucyte images of pHrodo‐labeled Aβ uptake in BV2 cells treated with vehicle or a PPAR‐γ agonist at 0 h and 1.5 h; (G) Quantification of the phagocytosis rate in BV2 cells under the indicated agonist or inhibitor treatments, expressed as the percentage of pHrodo‐positive area relative to total cell area ( n = 3); (H) qPCR analysis of Lilrb4a and Pparg expression in BV2 cells treated with vehicle or a PPAR‐γ agonist ( n = 3); (I) Schematic diagram of the FAM‐labeled Aβ phagocytosis and degradation assays in BV2 cells; (J) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis and after 3 h loading followed by 24 h degradation under the indicated PPAR‐γ agonist or inhibitor conditions; (K) Quantification of mean intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis (left) and after 24 h degradation (right) ( n = 3); (L) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in sorted primary microglia from LPS‐treated APOE4 mice under the indicated ASO and PPAR‐γ inhibitor conditions. Left, phagocytosis assay, in which cells were incubated with FAM‐Aβ and analyzed after 1.5 h to assess uptake. Right, degradation assay, in which cells were first allowed to internalize FAM‐Aβ for 3 h, then switched to substrate‐free medium, and analyzed 24 h later to assess residual intracellular signal; (M) Quantification of mean intracellular Alexa‐488 fluorescence in APOE4 microglia in the phagocytosis (left) and degradation (right) assays ( n = 3); (N,O) Corresponding representative histograms and quantification for sorted primary microglia from LPS‐treated APOE3 mice under identical assay conditions and treatment paradigms, with phagocytosis measured at 1.5 h and degradation measured 24 h after 3 h loading with FAM‐Aβ ( n = 3); (P) Representative immunofluorescence images of WT primary neuron–microglia co‐cultures treated with vehicle‐ or PPAR‐γ agonist‐treated microglia, showing DAPI (blue), MAP2 (green), and IBA1 (red) (10×, scale bar = 200 µm); (Q) Quantification of neuronal survival in co‐culture, measured as MAP2‐positive area ( n = 8–11). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two‐tailed Student's t test (C, E, H, and Q) or one‐way ANOVA (G, K, M, and O), as appropriate. ns, not significant. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: RNA Sequencing, Activation Assay, Biomarker Discovery, RNAscope, Staining, Labeling, Expressing, Flow Cytometry, Fluorescence, Phagocytosis Assay, Incubation, Degradation Assay, Immunofluorescence, Co-Culture Assay, Two Tailed Test