Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Determination of the interaction affinity between GM1 and APOE. A , schematic illustration of determining the binding affinity between lipid structure and APOE using MST. B , the binding affinity (K D ) between APOE3 or APOE4 and lipid structures containing GM1 or cholesterol (n = 3). C , the binding affinity between APOE3 or APOE4 and lipid structures with different GM1 concentrations. (n = 3). D - H , negative staining images of lipid structures with varying GM1 concentrations. 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 . APOE, apolipoprotein E; MST, microscale thermophoresis.

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, Negative Staining, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Analysis of GM1 localization and levels following the cellular uptake of lipid structures. A , schematic illustration of the method used to determine GM1 following the cellular uptake. B , GM1 levels in differentiated PC-12 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 3) ( C ) representative images showing GM1 and APOE localization in differentiated PC-12 cells following cellular uptake, as determined by confocal microscopy. (n ≥ 6) ( D ) GM1 levels in HEK-293 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 6) ( E ) (1) cellular uptake of DiD-labeled cholesterol and GM1 lipid structures in U-87 MG cells, and (2) changes in cholesterol levels in U-87 MG cells after cellular uptake of APOE3 and APOE4-enriched cholesterol lipoproteins, as well as a mixture of APOE3 and APOE4-enriched cholesterol and GM1 lipoproteins. (n ≥ 6). 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 . Created in BioRender. Dokholyan, N. (2025) https://BioRender.com/g17z389 . APOE, apolipoprotein E; HEK, human embryonic kidney; PC, l -α-phosphatidylcholine.

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: Confocal Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: The binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. A and B , the binding affinity between APOE3-enriched lipoprotein with varying GM1 concentration to LDLR. (n = 3) ( C and D ) the binding affinity between APOE4-enriched lipoprotein varying GM1 concentration to LDLR (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). APOE, apolipoprotein E; LDLR, low-density lipoprotein receptor; MST, microscale thermophoresis.

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, Concentration Assay, Microscale Thermophoresis

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 alters autophagic flux in in HEK293 cells stably expressing fluorescently tagged APOE. (A,C) HEK293 cells expressing APOE3–mCh or APOE4–mCh or mCh vector were analyzed by western blotting. (B) HEK293 cells stably expressing APOE3–mCh, APOE4–mCh, or mCh vector were treated with Baf and analyzed as for A and C (50 nM 4 h), Rap (10 nM 4 h) or both. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, ** P <0.01, **** P <0.0001 (one-way ANOVA with multiple comparisons correction).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE is turned over by autophagy in HEK293 cells with stable APOE expression. Representative images and fluorescence intensity of APOE3–mCh, APOE4–mCh, or mCh cells treated with Baf (50 nM 4 h), Epox (100 nM), or MG132 (50 µM). Quantitative results are mean±s.e.m. Bars over graphs indicate time points at which P <0.05 on two-way ANOVA with post-hoc Dunnett test.

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Expressing, Fluorescence

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 colocalizes with enlarged lysosomes. (A) HEK293 cells stably expressing APOE3– or APOE4–mCh (red) and transfected with CellLight Lamp1–GFP (green), and stained with DAPI (blue). (B) HEK293 cells stably expressing APOE3– or APOE4–mCh–SepH. SepH shown in green. (C) HEK293 cells expressing APOE2, APOE3, APOE4 or vector were treated with oleic acid (OA, overnight) and Baf (4 h). NT, no treatment. Quantitative results are mean±s.e.m. In A–C, three wells were imaged per APOE isoform (three images per well) by confocal microscopy. Images were analyzed using Imaris software. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 [Student's two-tailed unpaired t -test (A,B) or one-way ANOVA with a post-hoc Tukey–Kramer test (C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Transfection, Staining, Plasmid Preparation, Confocal Microscopy, Software, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE transiently overexpressed in ST14A cells is degraded by LAMP2A-dependent autophagy. (A) Schematic of dual-tag fluorescent APOE with quenching of green SepHluorin in lysosomes. (B) APOE3–mCh–SepH and mCh–SepH tag fluorescence intensity in ST14A cells with Baf (50 nM, 4 h). (C) APOE3 mRNA in ST14A cells expressing APOE3–Myc–flag with Baf treatment. (D) APOE3–Myc–Flag abundance in ST14A cells following Baf treatment (4 h 50 nM). (E) APOE3–mCh–SepH fluorescence intensity following BFA (5 μg/ml) treatment. (F) ST14A cells expressing APOE3-myc-flag and treated with BFA (5 μg/ml, 4 h) were analyzed by western blotting. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, *** P <0.001 [Student's unpaired two-tailed t -test was used (D); bars above graphs indicate time points at which FDR<0.05 by two-way ANOVA with post-hoc Tukey–Kramer test (B,E)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Expressing, Western Blot, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: LAMP2A is required for autophagy of APOE3 in ST14A cells. (A,B) ST14A cells were co-transfected with shRNA targeting LAMP2A and (A) APOE3–Myc–Flag or (B) APOE3–mCh, and APOE3 levels were assessed by western blot or fluorescence intensity. shCtrl, control shRNA. Bars above graph in B indicates time points at which FDR<0.05 by two-way ANOVA. (C) APOE levels in mouse brain tissue from 2-year-old wild-type and LAMP2 knockout mice. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, **** P <0.0001 [Student's two-tailed unpaired t -test (A,C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Transfection, shRNA, Western Blot, Fluorescence, Knock-Out, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Fluorescently tagged APOE is endocytosed in an isoform-dependent manner and alters endosomal morphology. (A,B) APOE–mCh-conditioned medium was collected from HEK293T cells and applied to (A) HepG2 and (B) ST14A cells. Cells were imaged and red fluorescence/phase area calculated. Bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA. (C) ST14A cells were treated for 24 h with conditioned medium with APOE3–mCh or APOE4–mCh (red), and immunocytochemistry for EEA1 or Rab7 (green) was performed, and cells stained with DAPI (blue). Cells were imaged by confocal microscopy and analyzed using Imaris image. Magnified view indicates enlarged images from white boxes in merge panel. (D) HepG2 cells were treated for 24 h with APOE3–mCh or APOE4–mCh conditioned medium, lysosomes were immunoprecipitated and proteomic contents analyzed by mass spectrometry. Proteins reduced in APOE4 lysosomes that were not reduced in mCh-treated cells included mitochondrial proteins such as prohibitin. Western blot analysis for prohibitin was performed on lyso-depleted flow-through. Revert, protein stain. HepG2 cells treated with APOE-mCh conditioned medium were also analyzed by qPCR. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001; ns, not significant (one-way ANOVA with Tukey–Kramer test).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Immunocytochemistry, Staining, Confocal Microscopy, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Knockdown of Rubicon or ATG7 reduces APOE internalization. (A,B) ATG7 and Rubicon were knocked down using siRNA in HepG2 cells, and (A) conditioned medium with APOE3–mCh or (B) medium containing LDL-pHrodo was applied. siCtrl, control siRNA. (C,D) HepG2 cells with siRNA knockdown of (C) ATG7 or (D) Rubicon were analyzed by qPCR. (E) HepG2 cells were treated with inhibitors and APOE3–mCh conditioned medium. Concentrations of inhibitors used were: 50 nM Bafilomycin A1, 20 mM ammonium chloride. NT, no treatment. Cells were imaged and fluorescence quantified by incucyte. Imaging and fluorescence quantification by incucyte. Quantitative results are mean±s.e.m. **** P <0.0001 [Student's two-tailed unpaired t -test (C,D); bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA with a post-hoc Dunnett's test (A,B,E)]

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: APOE treatment had no effects on ECM production by fibroblasts. (a) WB analysis of COL I and FN levels after treatment of fibroblasts with PBS or exogenous APOE (0.25 μg/mL) for 24 hours. (b) Quantification of COL I and FN levels. No significant difference is detected using unpaired t test. For each timepoint, three different samples were quantified (n=3).

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques:

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: (a) WB analysis of levels of APOE, CTSD and SPP1 in macrophages cultured on tissue culture plates (negative control, NC) and PCL scaffolds (PCL) for 48 hours and quantification of their levels. ** indicates p < 0.01, unpaired t test. For each timepoint and each group, three different samples were quantified (n=3). (b) Immunofluorescence staining of APOE and CD68, CTSD and CD68, SPP1 and CD68 in macrophages cultured on tissue culture plates and PCL scaffolds.

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques: Cell Culture, Negative Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: APOE KO reducing profibrotic macrophages during vascular regeneration. UMAP of macrophages in regenerated aortas post graft implantation in WT and Apoe -/- rats, heatmap of C2 scores in the UMAP of macrophages, and the percentage of C2 cells in the macrophages at Day 30 (a) and Day 90 (b). (c) Immunofluorescence staining of CD68 and CTSD in regenerated aortas post graft implantation in WT and Apoe -/- rats at Day 30 and Day 90. (d) Quantification of CD68 and CTSD double positive cells. ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, five different samples from five different animals were quantified (n=5). (e) WB analysis of levels of APOE, CTSD and SPP1 in regenerated aortas post graft implantation in WT and Apoe -/- rats at Day 30 and Day 90. (f) Quantification of levels of APOE, CTSD and SPP1. ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, five different samples from five different animals were quantified (n=5). (g) WB analysis of levels of APOE, CTSD and SPP1 in macrophages from WT and Apoe -/- rats after their culture on PCL scaffolds for 48 hours. (h) Quantification of levels of APOE, CTSD and SPP1 in the macrophages. ** indicates p < 0.01, unpaired t test. For each timepoint and each group, three different samples were quantified (n=3). (i) Immunofluorescence staining of APOE and CD68, CTSD and CD68, SPP1 and CD68 in macrophages from WT and Apoe -/- rats after their culture on PCL scaffolds for 48 hours.

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: APOE/LRP1 interaction promoting profibrotic transition of macrophages during vascular regeneration. (a) Immunoprecipitation (IP) following mass spectrometry (MS) to identify the potential receptors of APOE on surfaces of macrophages. (b) Co-immunoprecipitation (Co-IP) to confirm interaction between APOE and LRP1. (c) Immunofluorescence staining of CD68 and LRP1 in regenerated aortas post graft implantation across different timepoints. (d) Immunofluorescence staining of APOE and LRP1 in macrophages from WT rats after 48 hours of culture on PCL scaffolds. (e) WB analysis of levels of LRP1, APOE, CTSD and SPP1 in macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 hours prior to treatment with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 hours. (h) Quantification of levels of LRP1, APOE, CTSD and SPP1 in macrophages. * indicates p < 0.05, ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, three different samples were quantified (n=3). (e) Flow cytometry of CTSD positive cells in macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 hours prior to treatment with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 hours and quantification of percentage of CTSD positive cells. * indicates p < 0.05, Tukey’s post-hoc test. For each timepoint and each group, three different samples were quantified (n=3).

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques: Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Cell Culture, Negative Control, shRNA, Flow Cytometry

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: Fibroblast proliferation supported by profibrotic macrophages via IGF-1 secretion. Dot plots (a) and box plots (b) of Igf1 expression in macrophages of regenerated aortas post graft implantation in WT and Apoe -/- rats on Day 30 and Day 90. (c) Quantification of IGF-1 by ELISA in regenerated aortas post graft implantation in WT and Apoe -/- rats on Day 30 and Day 90. ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, five different samples from five different animals were quantified (n=5). UMAP of fibroblasts in regenerated aortas post graft implantation in WT and Apoe -/- rats, heatmap of cell cycle scores in the UMAP of fibroblasts, and box plots of cell cycle scores in the fibroblasts on Day 30 (d) and Day 90 (e). ** indicates p < 0.01, unpaired t test. (f) Immunofluorescence staining of Ki67 in regenerated aortas post graft implantation in WT and Apoe -/- rats on Day 30 and Day 90. L indicates lumens. (g) Quantification of Ki67 positive cells. ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, five different samples from five different animals were quantified (n=5). (h) Quantification of IGF-1 by ELISA in cell culture mediums after macrophages from WT or Apoe -/- rats were cultured on PCL scaffolds for 48 hours. ** indicates p < 0.01, unpaired t test. For each timepoint and each group, three different samples were quantified (n=3). (i) Quantification of proliferation of fibroblasts treated with condition medium (CM) with or without IGF-1 blocking antibody (Ab, 1 µg/mL) for 24 hours using cell counting kit-8 (CCK-8). ** indicates p < 0.01, Tukey’s post-hoc test. For each timepoint and each group, three different samples from three different animals were quantified (n=3).

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Cell Culture, Blocking Assay, Cell Counting, CCK-8 Assay

Journal: bioRxiv

Article Title: APOE knockout attenuates vascular graft fibrosis by limiting profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1101/2025.07.22.666232

Figure Lengend Snippet: (a) Macroscopic views, in vivo imaging system (IVIS), and immunofluorescence imaging of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe). (b) Quantification of transfection efficiency of regenerated aortas by AAV according to mScarlet expression. ** indicates p < 0.01, Tukey’s post-hoc test. For each group, six different images from six different animals were quantified (n=6). (c) Ultrasound imaging of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe). (d) Immunofluorescence staining of IGF-1 and CD68 in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe). (e) Quantification of IGF-1 positive areas in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe). ** indicates p < 0.01, Tukey’s post-hoc test. For each group, six different samples from six different animals were quantified (n=6).

Article Snippet: Exogenous APOE (MCE, HY-P701096), conditioned medium by macrophages, or IGF-1 blocking antibody (Invitrogen, MA5-18035) was added into culture medium as indicated.

Techniques: In Vivo Imaging, Immunofluorescence, Imaging, shRNA, Transfection, Expressing, Staining