3 h cholesterol  (Revvity)

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: HDL Nanodiscs Loaded with Liver X Receptor Agonist Decreases Tumor Burden and Mediates Long‐term Survival in Mouse Glioma Model

doi: 10.1002/smll.202307097

Figure Lengend Snippet: Efficacy of free LXR agonists and LXR‐CpG‐sHDL nanodiscs in GL26 cells. Changes in mRNA levels of A) LXRα, b) LXRβ, and C) ABCA1 following treatment with 5 µ m free or sHDL‐loaded LXR agonists evaluated via qPCR. D) Changes in protein expression evaluated via western blot. E) Radiolabeled cholesterol efflux from GL261 cells following 24‐hour treatment with 1 µ m LXR agonist or sHDL formulation. Viability of GL261 cells at increasing concentrations of LXR‐CpG‐sHDL formulations. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001. N = 3, Mean ± SEM.

Article Snippet: Cholesterol [1,2‐ 3 H(N)] was purchased from American Radiolabeled Chemicals (Saint Louis, MO).

Techniques: Expressing, Western Blot, Formulation

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Bulk-RNA and single-nucleus RNA (sn-RNA) sequencing analyses reveales an association between ABCA1 and cellular senescence in APOE4 and AD. ( A ) Bulk-RNA sequencing analysis showing cellular senescence ssGSEA scores across no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD. ( B ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores by Braak stage groups (0–3; 4; 5–6). ( C ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores in the groups with and without neuritic plaques. ( D ) Bulk-RNA sequencing analysis of cellular senescence ssGSEA scores across different APOE genotypes and clinical groups. ( E ) Bulk-RNA sequencing analysis of ABCA1 expression levels across clinical groups. ( F ) Association between ABCA1 expression and cellular senescence ssGSEA scores in the bulk-RNA sequencing, analyzed using a linear model. ( G ) Single-nucleus RNA sequencing analysis of the association of cellular senescence ssGSEA scores in different brain cells with factors, including AD pathology, sex, APOE genotype, LSX/RXR co-expression genes and other AD risk genes. The heatmap shows t value (coefficient 𝛃 divided by standard error of 𝛃), with red indicating significant positive associations, blue indicates significant negative associations, gray indicating non-significant associations, and cutoff p = 0.1). The gray bar plot represents ABCA1 expression levels in different cell types. ( H ) total cholesterol, ( I ) 7-hydroxycholesterol levels of the postmortem human cortex tissues from participants with or without AD and carrying different APOE genotypes. ( J ) Correlation between 7-hydroxycholesterol levels and cellular senescence ssGSEA scores. Data were analyzed using an unpaired t-test ( C ) or one-way ANOVA ( A , B , D , E ) or a linear model ( F ). ** p < 0.01, *** p < 0.01

Article Snippet: The cells were transfected with 20nM siRNA for 24 h, followed by labeling with 1μCi/mL (3 H) cholesterol (Moravek, MT9112) using serum-free DMEM/F12 containing 2 mg/ml fatty acid-free BSA (Sigma-Aldrich, catalog #A9647), and 2 μg/ml acyl-coenzymeA: cholesterol acyltransferase inhibitor SANDOZ (Sigma-Aldrich, catalog #S9318) for 24 h. After washing once with serum-free culture medium, the cells were treated with recombinant CS-6253 peptide (1 μM in DMEM/F12 containing 2 mg/mL fatty acid-free BSA, 2 μg/mL SANDOZ, and 200 μL/well) for 4 h. After treatment, the cell culture medium was collected and transferred to scintillation vials filled with 3 mL of the scintillation mixture.

Techniques: RNA Sequencing, Expressing

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Caveolin-1 regulates ABCA1 trafficking in cells and its expression is increased in APOE4 and AD biospecimens. ( A ) Schematic overview of the workflow for purification of the ABCA1 complex. ( B ) Co-immunoprecipitation of ABCA1 in ABCA1-GFP expressing and wild-type HeLa cells. ( C ) Co-immunoprecipitation in lysates of immortalized astrocytes using an ABCA1 antibody or species-matched IgG. ABCA1, Caveolin-1 and AP2B1 were detected by immunoprecipitation. ( D ) Co-staining for ABCA1 and caveolin-1 in immortalized astrocytes. Arrow indicates the cytoplasm, and arrowhead indicates the plasma membrane. ( E ) Co-staining for ABCA1 and AP2B1 in immortalized astrocytes. ( F ) Mouse primary astrocytes were transfected with non-target (NT), caveolin-1, and AP2B1 siRNA for 48 h. Plasma membrane protein was enriched by biotin agarose beads and the ABCA1 protein levels were detected by WB. Quantification was performed from two–three independent experiments. ( G ) Immortalized astrocytes were transfected with non-target or caveolin-1 siRNA for 24 h, followed by labeling with 3 H-cholesterol for 18 h. Cholesterol efflux was measured after treatment with recombinant ApoA1 or ApoE for 4 h ( n = 3 biological replicates). ( H ) Filipin staining of immortalized astrocytes loaded with or without low-density lipoprotein (LDL) (10 µg/mL) for 24 h. ( n = 7–12 random areas from two cultured wells). ( I ) Caveolin-1 protein levels in immortalized astrocytes loaded with or without LDL (10 µg/mL) for 24 h were detected by WB ( n = 3 biological replicates). ( J ) Protein levels of caveolin-1 and ABCA1 in the cortex of 8-months-old and 18-months-old APOE3 and APOE4-TR mice ( n = 5–6 mice for each genotype, mixed gender). ( K ) For 22-months-old APOE3 and APOE4-TR mice, the cortex was sequentially homogenate with TBS, TBSX and GnHCl. ABCA1 and caveolin-1 protein levels in TBSX-soluble (membrane enriched) and GnHCl-soluble (aggregated protein enriched) fractions were measured by WB. ( n = 7 mice for each genotype, both sexes). (L) Protein levels of caveolin-1 and ABCA1 in the cortex of 6-months-old APP/PS1/APOE3 and APP/PS1/APOE4 mice were detected by WB ( n = 6 mice of each genotype, both sexes). ( M ) Total membrane caveolin-1 protein levels in human postmortem mid-frontal lobe tissues from reactive oxygen species (ROS) were detected by WB. Quantification of the relative intensity of caveolin-1 normalized to the plasma membrane marker Na, K-ATPase (NCI APOE3/3, n = 29; AD APOE3/3, n = 44; NCI APOE3/4, n = 19; AD APOE3/4, n = 42). Data are represented as mean ± SD and analyzed using two-tailed t-test ( F , G , H , I , J , K and L ) or one-way ANOVA followed by Tukey’s test ( M ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The cells were transfected with 20nM siRNA for 24 h, followed by labeling with 1μCi/mL (3 H) cholesterol (Moravek, MT9112) using serum-free DMEM/F12 containing 2 mg/ml fatty acid-free BSA (Sigma-Aldrich, catalog #A9647), and 2 μg/ml acyl-coenzymeA: cholesterol acyltransferase inhibitor SANDOZ (Sigma-Aldrich, catalog #S9318) for 24 h. After washing once with serum-free culture medium, the cells were treated with recombinant CS-6253 peptide (1 μM in DMEM/F12 containing 2 mg/mL fatty acid-free BSA, 2 μg/mL SANDOZ, and 200 μL/well) for 4 h. After treatment, the cell culture medium was collected and transferred to scintillation vials filled with 3 mL of the scintillation mixture.

Techniques: Expressing, Purification, Immunoprecipitation, Staining, Clinical Proteomics, Membrane, Transfection, Labeling, Recombinant, Cell Culture, Marker, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Reduction of oxysterols by HPCD increases ABCA1 and endo-lysosome recycling in APOE4-TR mice. ( A ) Study design and timeline for mouse experiments. APOE4-TR mice were injected with (2-Hydroxypropyl)-β-cyclodextrin (HPCD) for 2 months, followed by behavioral and pathological tests ( n = 14, 5 females and 9 males for PBS group; n = 15, 5 females and 10 males for the HPCD group). ( B ) Total cholesterol levels in mouse brain homogenates. ( C-E ) Oxysterols including 7β-hydroxycholesterol ( C ), 25-hydroxycholesterol ( D ) and 7α,25-dihydroxycholesterol ( E ) levels in mouse brain were measured by mass spectrometry. ( F ) Total membrane protein levels of ABCA1 and caveolin-1 in the mouse cortex detected by WB. ( G ) Lysosomes were isolated from mouse brains. ABCA1 protein levels in lysosomes were detected by WB. ( H ) Representative confocal images of LAMP1 and ABCA1 staining in mouse brain sections. Quantification of ABCA1 intensity in LAMP1 + area. ( n = 150 LAMP1 + ROI from 5 mice in each group; A.U.). ( I ) Protein levels of EEA1 (early endosome marker), Rab9 (late endosome marker), and LAMP1 (lysosome marker) in the membrane fraction (TBSX) and the intracellular fraction (GnHCl) were detected by WB. ( n = 10, five females and five males in each group for B-G, and I). Data are represented as mean ± SD and analyzed using a two-tailed t-test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001. Females are indicated with empty dots and males are indicated with filled dots for all data plots

Article Snippet: The cells were transfected with 20nM siRNA for 24 h, followed by labeling with 1μCi/mL (3 H) cholesterol (Moravek, MT9112) using serum-free DMEM/F12 containing 2 mg/ml fatty acid-free BSA (Sigma-Aldrich, catalog #A9647), and 2 μg/ml acyl-coenzymeA: cholesterol acyltransferase inhibitor SANDOZ (Sigma-Aldrich, catalog #S9318) for 24 h. After washing once with serum-free culture medium, the cells were treated with recombinant CS-6253 peptide (1 μM in DMEM/F12 containing 2 mg/mL fatty acid-free BSA, 2 μg/mL SANDOZ, and 200 μL/well) for 4 h. After treatment, the cell culture medium was collected and transferred to scintillation vials filled with 3 mL of the scintillation mixture.

Techniques: Injection, Mass Spectrometry, Membrane, Isolation, Staining, Marker, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Cellular senescence induced by cholesterol accumulation is mediated by lysosomal ABCA1 in APOE4 and AD

doi: 10.1186/s13024-025-00802-7

Figure Lengend Snippet: Oxysterol accumulation promotes cellular senescence and neuroinflammation in APOE4 and AD through the aggregation of ABCA1 in lysosomes. Compared to healthy cells, a higher oxysterol burden in AD, particularly in APOE4 carriers, leads to increased ABCA1 and caveolin-1 expression, and lysosomal dysfunction marked by increased lipofuscin staining. This sequestration reduces ABCA1 endosome-lysosome recycling, and exacerbates cholesterol and lipid accumulation within lysosomes, activating mTORC1, and contributing to cellular senescence and neuroinflammation. Treatment with cyclodextrin mitigates these effects by lowering oxysterols. Illumination is created by BioRender. CD: Cyclodextrin

Article Snippet: The cells were transfected with 20nM siRNA for 24 h, followed by labeling with 1μCi/mL (3 H) cholesterol (Moravek, MT9112) using serum-free DMEM/F12 containing 2 mg/ml fatty acid-free BSA (Sigma-Aldrich, catalog #A9647), and 2 μg/ml acyl-coenzymeA: cholesterol acyltransferase inhibitor SANDOZ (Sigma-Aldrich, catalog #S9318) for 24 h. After washing once with serum-free culture medium, the cells were treated with recombinant CS-6253 peptide (1 μM in DMEM/F12 containing 2 mg/mL fatty acid-free BSA, 2 μg/mL SANDOZ, and 200 μL/well) for 4 h. After treatment, the cell culture medium was collected and transferred to scintillation vials filled with 3 mL of the scintillation mixture.

Techniques: Expressing, Staining