Journal: Journal of Lipid Research

Article Title: Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice

doi: 10.1194/jlr.m800542-jlr200

Figure Lengend Snippet: Fig. 2. Effects of overexpressed human PCSK9 on LDLR protein levels in transgenic mice fed with chow diet. A: hPCSK9 expression markedly diminished liver LDLR proteins. Liver extracts from chow fed mice were pooled for immunoblot analysis. n = 3 for each group. B: hPCSK9 expression reduced kidney LDLR protein levels and did not alter adrenal LDLR proteins. Tissue extracts were pooled from chow diet fed wild-type and hPCSK9 trangenic mice as described in Materials and Methods and were subjected to immu- noblot analysis. n = 3 for each group. LDLR, LDL receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: Mouse LDLR protein was detected with a goat anti-mouse LDLR antibody (R and D Systems Cat# AF2255), and human PCSK9 protein in transgenic mice was detected using antibody against V5-tag (Sigma). joined with transgenic mice ( 18 ).

Techniques: Transgenic Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Human LDLR is proteolytically cleaved in its extracellular ligand binding domain by BMP1. ( A ) Schematic of the domain organisation of LDLR with a C-terminal FLAG tag showing the epitopes detected by the antibodies used in the study, antibody AF2148 (R&D Systems) raised against the entire ectodomain of LDLR, antibody Ab14056 (Abcam) raised against a recombinant protein fragment corresponding to amino acids 29–205 of LDLR) and antibody α-FLAG (Sigma-Aldrich) the anti-FLAG M2 antibody. TM, transmembrane domain; EGF, epidermal growth factor-like domain; F, FLAG epitope. ( B ) Immunoblot analysis with the indicated antibody of lysates and conditioned media samples from HepG2 cells expressing full-length FLAG-tagged human LDLR. Bands of interest were cropped from western blots of either media or lysate samples using each of the three antibodies. Images from separate western blots were combined but are separated by the dashed black line. Full blot images are presented in the Supplementary western blot dataset. ( C ) Schematic showing the proposed cleavage of the 160 kDa full-length (FL) LDLR to generate the 36–40 kDa NTF and 120 kDa CTF. ( D ) Immunoblot analysis of LDLR (antibody AF2148) without and with deglycosylation in liver biopsy samples from three separate individuals. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. ( E ) Immunoblot analysis following incubation of rhLDLR (500 ng) with increasing amounts of rBMP1 at 37 °C for 1 h. ( F ) Immunoblot analysis following incubation of rhLDLR (500 ng) with rBMP1 (12.5 ng) in the absence or presence of the BMP1 inhibitor UK383367 (10 μM) at 37 °C for 1 h. ( G ) Immunoblot analysis following pre-incubation of rhLDLR (500 ng) in the absence or presence of LDL (5 µg), RAP (7.14pmol) or UK383367 (10 μM) for 30 min on ice followed by the addition of 12.5 ng rBMP1 and further incubation at 37 °C for 1 h. ( H ) Densitometric analysis of the Ab14056 immunoblot from ( C ) to determine the amount of FL and NTF as a percentage of total LDLR, data shown as mean ± SEM, statistical analysis using ANOVA with Tukey post-hoc pairwise analysis *p < 0.05, n = 3. For panels E–G, blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL and NTF bands using the Ab14056 antibody due to different exposure times for visualisation of the FL and NTF bands. Full blot images are presented in the Supplementary western blot dataset.

Article Snippet: Recombinant human LDLR (rhLDLR; 500 ng; 100 ng/μl) (R&D systems, Abingdon, UK) was incubated with increasing concentrations (0 to 12.5 ng) of recombinant BMP1 (rBMP1) (R&D Systems) in HEPES buffer (25 mM HEPES, 0.01% Brij 35, pH 7.5) in a total volume of 15 μl for 1 h at 37 °C.

Techniques: Ligand Binding Assay, FLAG-tag, Recombinant, Western Blot, Expressing, Incubation

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Cleavage of human LDLR by BMP1 reduces the binding and cellular uptake of LDL. ( A ) Immunoblot analysis following incubation of rhLDLR-His (5 μg) with rBMP1 (200 ng) at 37 °C for 1 h. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. ( B ) Dot blot of BODIPY ® -LDL (1 μg/ml) binding to rhLDLR-His digested with BMP1 (from A), BACE1-His and PCSK9-FLAG (all 0.5 μg). Membranes were re-probed with anti-His and anti-FLAG antibodies. ( C ) Densitometric analysis of the BODIPY-LDL dot blot from ( B ) showing the percentage binding to rhLDLR-His in the absence or presence of rBMP1. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 4. ( D ) Immunoblot analysis of LDLR with actin as a loading control in lysates from CHO-A7 cells expressing either full-length (FL) LDLR or 120 kDa CTF, or the empty vector (EV). The blot images were cropped to highlight the FL and CTF bands using the AF2148 and anti-FLAG antibodies and actin using the AC15 antibody. The full blot images are presented in the Supplementary western blot dataset. ( E ) Immunoblot analysis of LDLR from lysates (L) and from the cell-surface fraction (S) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with either vehicle (DMSO) or EZ-Link™ Sulfo-NHS-SS-Biotin (0.5 mg/ml) for 20 min on ice and immunoprecipitation with Streptavidin-agarose beads for 3 h at 4 °C. The blot image was cropped to highlight the FL and CTF bands using the AF2148 antibody. The full blot image is presented in the Supplementary western blot dataset. ( F ) Immunofluorescence microscopy images showing LDLR expression (green) and Dil-LDL binding (red) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with Dil-LDL for 30 s on ice. ( G ) Quantification of Dil-LDL mean intensity in either the FL LDLR or 120 kDa CTF expressing cells from ( F ), data shown as mean ± SEM, statistical analysis performed using a Student’s T-test *p < 0.05, n = 7. ( H ) BODIPY ® -LDL uptake in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF. Data expressed as percentage of FL following subtraction of empty vector uptake as a baseline and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, *p < 0.05, n = 4.

Article Snippet: Recombinant human LDLR (rhLDLR; 500 ng; 100 ng/μl) (R&D systems, Abingdon, UK) was incubated with increasing concentrations (0 to 12.5 ng) of recombinant BMP1 (rBMP1) (R&D Systems) in HEPES buffer (25 mM HEPES, 0.01% Brij 35, pH 7.5) in a total volume of 15 μl for 1 h at 37 °C.

Techniques: Binding Assay, Western Blot, Incubation, Dot Blot, Control, Expressing, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Genetic knockdown of BMP1 decreases the proteolytic cleavage of LDLR. ( A ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Dharmacon) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of the NTF from ( A ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 6. ( C ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( D ) Relative expression of BMP1 mRNA in HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control. Data expressed relative to control and shown as mean ± SEM, statistical analysis performed using an independent t-test with Welch’s correction for two sample comparison **p < 0.01, n = 3. ( E ) Immunoblot analysis of LDLR and BMP1 in cell lysates from HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA and then transfected either with an empty vector, BMP1-FLAG or BMP1 E 214 Q-FLAG constructs. Blot images were cropped to highlight the FL and CTF band of LDLR using the AF2148 antibody and BMP1 using the AF1927 antibody. Full blot images are presented in the Supplementary western blot dataset. ( F ) Densitometric analysis of FL LDLR and the CTF from ( E ). Data expressed as percentage of NT siRNA control (not shown in graph) and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, ***p < 0.001, n = 3.

Article Snippet: Recombinant human LDLR (rhLDLR; 500 ng; 100 ng/μl) (R&D systems, Abingdon, UK) was incubated with increasing concentrations (0 to 12.5 ng) of recombinant BMP1 (rBMP1) (R&D Systems) in HEPES buffer (25 mM HEPES, 0.01% Brij 35, pH 7.5) in a total volume of 15 μl for 1 h at 37 °C.

Techniques: Knockdown, Western Blot, Control, Expressing, Comparison, Transfection, Plasmid Preparation, Construct

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Pharmacological inhibition of BMP1 decreases the proteolytic cleavage of LDLR and increases the cellular uptake of LDL. ( A ) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated with inhibitor UK383367 (10 μM) for 18 h. Blot images were cropped to highlight the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of the NTF from ( A ). Data shown as mean ± SEM, ***p < 0.001, n = 3. ( C ) BODIPY ® -LDL uptake in HepG2 cells treated with or without inhibitor UK383367 (10 μM) for 6 h. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test **p < 0.01, n = 3. ( D ) Representative data from flow cytometry showing the change in cell surface fluorescence of LDLR between HepG2 cells treated with inhibitor UK383367 (10 μM) for 18 h (blue line) and untreated cells (red line); primary antibody (Ab14056) only (grey line). ( E ) Quantitation of the cell surface expression of LDLR in HepG2 cells treated with or without UK383367 as analysed by flow cytometry in ( D ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test **p < 0.01, n = 3.

Article Snippet: Recombinant human LDLR (rhLDLR; 500 ng; 100 ng/μl) (R&D systems, Abingdon, UK) was incubated with increasing concentrations (0 to 12.5 ng) of recombinant BMP1 (rBMP1) (R&D Systems) in HEPES buffer (25 mM HEPES, 0.01% Brij 35, pH 7.5) in a total volume of 15 μl for 1 h at 37 °C.

Techniques: Inhibition, Western Blot, Control, Flow Cytometry, Fluorescence, Quantitation Assay, Expressing

Journal: Scientific Reports

Article Title: Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

doi: 10.1038/s41598-019-47814-0

Figure Lengend Snippet: Cleavage of LDLR requires the catalytic activity of BMP1 and human but not murine LDLR is proteolytically cleaved by BMP1 in vivo . ( A ) Immunoblot analysis of BMP1 in conditioned media and of LDLR in cell lysates and conditioned media from SW13 cells expressing wild-type (WT) or mutated (E 214 Q) BMP1. Blot images were cropped to highlight BMP-1 bands in conditioned media and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. ( B ) Densitometric analysis of FL and the NTF from ( A ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis *p < 0.05, **p < 0.01, n = 4. ( C ) BODIPY ® -LDL uptake in SW13 cells expressing either WT or E 214 Q BMP1. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, n = 3. ( D ) Immunoblot analysis of rhLDLR (500 ng) (top panel) and recombinant mouse LDLR (500 ng) (bottom panel) following incubation with rBMP1 (100 ng) for 1 h. Blot images were cropped to highlight the FL and CTF bands in human LDLR using the AF2148 antibody and the FL band only in mouse LDLR using AF2255 antibody. Full blots are not available for this figure. ( E ) Immunoblot analysis of LDLR in lysates from HepG2 cells expressing either wild-type (WT) or mutated (D 172 V) LDLR and incubated with or without inhibitor UK383367 (10 μM) for 18 h. The blot image was cropped to highlight the FL and CTF bands using the anti-FLAG antibody. Full blot images are presented in the Supplementary western blot dataset. ( F ) Densitometric analysis of FL -LDLR and the 120 kDa CTF from ( E ). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, n = 3. ( G ) Immunoblot analysis of human LDLR (top panel) and mouse LDLR (bottom panel) in liver lysates from either wild-type LDLR +/+ or transgenic LDLR h/+ mice. Blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL band only in mouse LDLR using AF2255 antibody. Full blot images are presented in the Supplementary western blot dataset.

Article Snippet: Recombinant human LDLR (rhLDLR; 500 ng; 100 ng/μl) (R&D systems, Abingdon, UK) was incubated with increasing concentrations (0 to 12.5 ng) of recombinant BMP1 (rBMP1) (R&D Systems) in HEPES buffer (25 mM HEPES, 0.01% Brij 35, pH 7.5) in a total volume of 15 μl for 1 h at 37 °C.

Techniques: Activity Assay, In Vivo, Western Blot, Expressing, Control, Recombinant, Incubation, Transgenic Assay

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 1. ApoE isoform-dependent LDLR binding results in differential cellular uptake (A and B) Confocal images and quantification of FITC-labeled POPC-lipidated ApoE (10 mg/mL) bound to surface of 293T cells overexpressing LDLR after 1 h incubation at 4C. Scale bar: 10 mm in (A). (C) HTRF showing POPC-lipidated ApoE binding to LDLR ECD (3 independent experiments). (D) SPR profiles for POPC-lipidated ApoE isoform binding to biotinylated LDLR ECD. Red: experimental data. Black: curve fit using a 1:1 kinetic binding model. Each curve represents binding at one concentration (0.5, 1, and 2 mM for lipApoE2; 0.0156, 0.03125, and 0.0625 mM for lipApoE3/E4).

Article Snippet: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

Techniques: Binding Assay, Labeling, Incubation, Concentration Assay

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 4. Differential lipid burden modulates inflammatory responses and transcription of microglia (A and B) Confocal images and quantification of BODIPY signals in APOE KO iMg after 1-day incubation with 10 mg/mL BODIPY-CE pre-complexed with 10 mg/mL HDL and 10 mg/mL ApoE, with and without co-treatment of 20 mg/mL LDLR ECD, in medium containing 100 ng/mL LPS. Scale bar: 20 mm for (A). (C) Relative expression of 5 genes quantified by qPCR for APOE KO iMg after live imaging shown in (A).

Article Snippet: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

Techniques: Incubation, Expressing, Imaging

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 7. Christchurch mutation reduces LDLR binding, lipid uptake, and lipofuscin (A) Competitive HTRF demonstrating inhibition of tagged lipApoE4-LDLR ECD binding in the presence of different concentrations of untagged lipidated ApoE variants (n = 3 independent experiments). No inhibitor: no addition of untagged lipidated ApoE. The first panel shows the complete dose response. The second and third panels show the % inhibition at 125 and 250 nM, respectively. (B and C) Confocal images and quantification of pHrodo green-labeled POPC-lipidated ApoE isoforms (10 mg/mL) in H4 cells after 1-day incubation. Scale bar: 10 mm in (B). (D and E) Confocal images and quantification of pHrodo signals detected in H4 cells after 1-day incubation with 10 mg/mL pHrodo green-labeled HDL pre- complexed with 10 mg/mL ApoE isoforms. Scale bar: 10 mm in (D). (F and G) Confocal images and quantification of lipofuscin in H4 cells after 3-day incubation with 20 mg/mL CE(20:4)/POPC-lipidated ApoE isoforms. Scale bar: 10 mm in (F). In all bar graphs, data shown are mean + SEM with each dot representing one independent experiment; **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S7.

Article Snippet: To test how uptake of lipApoE is mediated by cell surface receptors, pHrodo green-lipApoE was pre-incubated with LDLR ECD (R&D Systems #2148-LD/CF-MTO, or generated as described above), low-molecular-weight heparin (Fisher Scientific, BP2524), or CD98hc ECD84 at 37 C for 30–40 min before being added to cells.

Techniques: Mutagenesis, Binding Assay, Inhibition, Labeling, Incubation

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 1. ApoE isoform-dependent LDLR binding results in differential cellular uptake (A and B) Confocal images and quantification of FITC-labeled POPC-lipidated ApoE (10 mg/mL) bound to surface of 293T cells overexpressing LDLR after 1 h incubation at 4C. Scale bar: 10 mm in (A). (C) HTRF showing POPC-lipidated ApoE binding to LDLR ECD (3 independent experiments). (D) SPR profiles for POPC-lipidated ApoE isoform binding to biotinylated LDLR ECD. Red: experimental data. Black: curve fit using a 1:1 kinetic binding model. Each curve represents binding at one concentration (0.5, 1, and 2 mM for lipApoE2; 0.0156, 0.03125, and 0.0625 mM for lipApoE3/E4).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

Techniques: Binding Assay, Labeling, Incubation, Concentration Assay

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 4. Differential lipid burden modulates inflammatory responses and transcription of microglia (A and B) Confocal images and quantification of BODIPY signals in APOE KO iMg after 1-day incubation with 10 mg/mL BODIPY-CE pre-complexed with 10 mg/mL HDL and 10 mg/mL ApoE, with and without co-treatment of 20 mg/mL LDLR ECD, in medium containing 100 ng/mL LPS. Scale bar: 20 mm for (A). (C) Relative expression of 5 genes quantified by qPCR for APOE KO iMg after live imaging shown in (A).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

Techniques: Incubation, Expressing, Imaging

Journal: Cell

Article Title: Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes.

doi: 10.1016/j.cell.2024.10.027

Figure Lengend Snippet: Figure 7. Christchurch mutation reduces LDLR binding, lipid uptake, and lipofuscin (A) Competitive HTRF demonstrating inhibition of tagged lipApoE4-LDLR ECD binding in the presence of different concentrations of untagged lipidated ApoE variants (n = 3 independent experiments). No inhibitor: no addition of untagged lipidated ApoE. The first panel shows the complete dose response. The second and third panels show the % inhibition at 125 and 250 nM, respectively. (B and C) Confocal images and quantification of pHrodo green-labeled POPC-lipidated ApoE isoforms (10 mg/mL) in H4 cells after 1-day incubation. Scale bar: 10 mm in (B). (D and E) Confocal images and quantification of pHrodo signals detected in H4 cells after 1-day incubation with 10 mg/mL pHrodo green-labeled HDL pre- complexed with 10 mg/mL ApoE isoforms. Scale bar: 10 mm in (D). (F and G) Confocal images and quantification of lipofuscin in H4 cells after 3-day incubation with 20 mg/mL CE(20:4)/POPC-lipidated ApoE isoforms. Scale bar: 10 mm in (F). In all bar graphs, data shown are mean + SEM with each dot representing one independent experiment; **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Goat anti-human LDLR R&D Systems Cat: AF2148; RRID:AB_2135126 Rabbit anti-human ApoE Abcam Cat: ab52607; RRID:AB_867704 Mouse pan anti-human ApoE Cell Signaling Cat: 74417; RRID:AB_3094529 Mouse anti-LAMP1 DSHB Clone H4A3; RRID:AB_2296838 Rabbit anti-PLIN2 (ADFP) ThermoFisher Scientific Cat: MA5-32664; RRID:AB_2809941 Mouse anti-TuJ1 (b3-tubulin) Cell Signaling Cat: 4466S; RRID:AB_1904176 Chicken anti-MAP2 (microtubule-associated protein 2) Abcam Cat: ab5392; RRID:AB_2138153 Mouse anti-4-HNE Abcam Cat: ab48506; RRID:AB_867452 AT8 (Mouse anti-pS202/T205 tau) ThermoFisher Scientific Cat: MN1020; RRID:AB_223647 Goat anti-Iba1 Novus Cat: NB100-1028; RRID:AB_3148646 or AB_521594 Rabbit anti-NeuN Abcam Cat: ab177487; RRID:AB_2532109 Donkey anti-mouse 488nm ThermoFisher Scientific Cat: A-21202 Donkey anti-rabbit 488nm ThermoFisher Scientific Cat: A-21206 Donkey anti-rabbit 488nm PLUS ThermoFisher Scientific Cat: A-32790 Donkey anti-rabbit 568nm ThermoFisher Scientific Cat: A-10042 Donkey anti-mouse 647 nm ThermoFisher Scientific Cat: A-31571 Donkey anti-rabbit 647nm ThermoFisher Scientific Cat: A-31573 Donkey anti-goat 647nm ThermoFisher Scientific Cat: A-21447 Donkey anti-chicken 647nm ThermoFisher Scientific Cat: A-78952 Biological samples Human CSF samples Precision Med 7005, 8009, and 8200 Chemicals, peptides, and recombinant proteins ApoE2, ApoE3, R136S ApoE3, ApoE4, R136S ApoE4, K146E ApoE4 Generated in-house N/A LDLR ECD R&D Systems; or generated in-house Cat: 2148-LD/CF-MTO Heparin Fisher Scientific Cat: BP2524 DiI-LDL ThermoFisher Scientific Cat: L3482 HDL EMD Millipore Cat: LP3-5MG BODIPY-CE ThermoFisher Scientific Cat: C3927MP CE(18:1) Avanti Polar Lipids Cat: 700269 CE(20:4) Cayman Chem Cat: 22595 POPC Avanti Polar Lipids Cat: 850457 PBS Gibco Cat: 10010023 Bio-Beads SM-2 Resin Bio-Rad Cat: 1523920 Lipofectamine LTX-Plus Invitrogen Cat: A12621 Bovine Serum Albumin Sigma-Aldrich Cat: A2153-500G NucBlue Live ReadyProbes ThermoFisher Scientific Cat: R37605 DAPI Invitrogen Cat: D1306 Fluorescein-transferrin ThermoFisher Scientific Cat: T2871 LysoTracker Deep Red Invitrogen Cat: L12492 InstantBlue Abcam Cat: ab119211 ProLong Gold Antifade Mountant ThermoFisher Scientific Cat: P36930 (Continued on next page) ll OPEN ACCESS Cell 188, 1–20.e1–e13, January 9, 2025 e1 Please cite this article in press as: Guo et al., Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes, Cell (2025), https://doi.org/10.1016/j.cell.2024.10.027 Article

Techniques: Mutagenesis, Binding Assay, Inhibition, Labeling, Incubation