Journal: International Journal of Molecular Sciences

Article Title: iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

doi: 10.3390/ijms25020689

Figure Lengend Snippet: iPSC-derived endothelium from patients with FH shows reduced LDL uptake capacity. ( a ) Representative images showing differences in the fluorescently labeled LDL uptake capacity (red signal) between endothelial derivatives obtained from iPSCs with normal (EC K7-4) and pathological (EC FH 1.3.1 and EC FH 3.2.8) LDLR allelic variants. Nuclei are stained with DAPI. The scale bar is 100 microns. Magnification 200×. The experiment was performed in triplicate. ( b ) Quantification of LDL uptake capacity for control and patient-specific IPSC-derived endothelial cells in 10 fields of view for each sample.

Article Snippet: Recombinant human LDLR (R&D Systems, NE Minneapolis, MN, USA) was used as a control.

Techniques: Derivative Assay, Labeling, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

doi: 10.3390/ijms25020689

Figure Lengend Snippet: LDLR protein expression in iPSCs and their endothelial derivatives from patients with FH and a healthy donor. ( a ) Immunoblotting revealed a decreased level of mature LDLR in iPSCs and iPSC-ECs from patients with FH (FH 1.3.1 and FH 3.2.8) and an increased level of immature LDLR in iPSCs and iPSC-ECs from FH 1.3.1 compared to a healthy donor (K7-4). ( b ) Relative densitometric quantification of total LDL (mature and immature forms together) in iPSCs and iPSC-ES from patients with FH (FH 1.3.1 and FH 3.2.8) and a healthy donor (K7-4). The experiment was performed in triplicate. ( c ) Relative densitometric quantification of mature and immature LDL in iPSCs and iPSC-ES from patients with FH (FH 1.3.1 and FH 3.2.8) and a healthy donor (K7-4). The experiment was performed in triplicate.

Article Snippet: Recombinant human LDLR (R&D Systems, NE Minneapolis, MN, USA) was used as a control.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

doi: 10.3390/ijms25020689

Figure Lengend Snippet: Transcriptional profiling of the control (CTRL) and LDLR mutant (FH) iPSC-ECs. ( a ) PCA plot for two control (CTRL) and two LDLR mutant (FH) iPSC-EC cultures. ( b ) Volcano plot of 39 differentially expressed genes (DEGs) between CTRL and FH. FDR, false discovery rate; FC, fold change. The top five DEGs are shown. ( c ) Heat map of 39 DEGs. The color of each dot represents the gene expression in the sample. The brighter the red, the higher the expression; the brighter the blue, the lower the expression. ( d ) Histograms showing gene ontology of biological processes and reactome gene sets with upregulated (logFC ≥ 1) and downregulated (logFC ≤ −1) expression in FH compared to CTRL. ( e ) Bioinformatic analysis of DEGs using STRING to identify functional interactions between deregulated proteins. Each node represents a protein, and each edge represents an interaction.

Article Snippet: Recombinant human LDLR (R&D Systems, NE Minneapolis, MN, USA) was used as a control.

Techniques: Control, Mutagenesis, Gene Expression, Expressing, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

doi: 10.3390/ijms25020689

Figure Lengend Snippet: Primary and secondary antibodies.

Article Snippet: Recombinant human LDLR (R&D Systems, NE Minneapolis, MN, USA) was used as a control.

Techniques: Immunofluorescence, Western Blot

Journal: Nature communications

Article Title: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

doi: 10.1038/s41467-021-22167-3

Figure Lengend Snippet: Fig. 1 MT1-MMP-mediated LDLR degradation. a Knockdown of MT1-MMP expression. Whole-cell lysate from Huh7.5 cells transfected with scrambled (Scram) or one of the two different MT1-MMP siRNAs (MT1-1, MT1-2) was applied to immunoblotting. TFR, transferrin receptor. b Effect of MT1-MMP knockdown on PCSK9-promoted LDLR degradation. Huh7.5 cells transfected with scrambled or MT1-MMP siRNA were incubated with or without PCSK9 (2 μg/ml). Whole-cell lysate was applied to western blot with antibodies indicated. c Effect of MT1-MMP knockdown in HepG2 cells. The cells were transfected with scrambled (Scram) or MT1-MMP siRNAs (MT1-1, MT1-2) for 48 h. Same amount of whole-cell lysate was applied to immunoblotting. The images showed representative protein levels. Similar results were obtained from three independent experiments. The relative densitometry was the ratio of the densitometry of LDLR to that of transferrin receptor (TFR) at the same condition (n = 3 independent experiments). The percentage of relative densitometry was the ratio of the relative densitometry of LDLR at different treatments to that of LDLR at the control condition, which was defined as 100%. d Effect of MT1-MMP overexpression on LDLR. Whole-cell lysate was isolated from Huh7.5 cells transfected with empty plasmid (Con) or different amount of plasmid carrying MT1-MMP cDNA, and then subjected to immunoblotting. e Biotinylation of cell surface proteins. Huh7.5 cells transfected with scrambled (Scram) or MT1-MMP siRNA (MT1-1) were biotinylated. Same amount of total proteins in whole-cell lysate was applied to NeutrAvidin beads to pull down cell surface proteins, followed by immunoblotting. Cal, calnexin. f LDL uptake (n = 3 independent experiments). Huh7.5 cells transfected with scrambled or MT1-MMP siRNA (MT1-1) were labeled with Dil-LDL in the absence and presence of unlabeled LDL. Fluorescence intensity (RFU) was measured to calculate specific binding. Similar results were obtained from at least three independent experiments. Student’s t test (two-sided) was carried out to determine the significant differences between groups (c and f). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file.

Article Snippet: The levels of total cholesterol and sLDLR in each fraction were measured using the Cholesterol E kit (Wako Diagnostics) and the Human or Mouse LDLR DuoSet ELISA assay kit (R&D system), respectively.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Incubation, Control, Over Expression, Isolation, Plasmid Preparation, Labeling, Fluorescence, Binding Assay

Journal: Nature communications

Article Title: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

doi: 10.1038/s41467-021-22167-3

Figure Lengend Snippet: Fig. 5 The impact of MT1-MMP. a Overexpression of human MT1-MMP. Male C57BL/6J mice were injected with empty or the wild-type MT1-MMP adenoviruses (5 mice per group). Liver homogenate was subject to immunoblotting, followed by quantification of LDLR levels relative to actin in the same mouse. Representative images were shown. Similar results were obtained from other mice. Plasma levels of HDL (b) and non-HDL cholesterol (c) (4 mice per group). d Effect of the Western-type diet (6 mice per group). Mice were fed the Western-type diet for 8 weeks. Liver homogenate was subjected to immunoblotting. Relative densitometry of LDLR was determined as described. Actin was used as a loading control. Representative images were shown. Similar results were obtained from the other mice. Liver section staining. Representative figures and quantification (ImageJ 1.52S) of Masson’s Trichrome (e) and oil Red-O staining (f) in cross-sections of the liver (6 mice per group). Similar results were obtained from the other mice (magnification: 400×). Plasma levels of cholesterol content in HDL (g) and non-HDL (h) (4 mice per group). i Lipid profile. Same amount of plasma from each mouse in the same group (2 female and 2 male mice per group) was pooled and applied to FPLC analysis of plasma cholesterol. Student’s t test (two-sided) was carried out to determine the significant differences between groups (a–h). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file.

Article Snippet: The levels of total cholesterol and sLDLR in each fraction were measured using the Cholesterol E kit (Wako Diagnostics) and the Human or Mouse LDLR DuoSet ELISA assay kit (R&D system), respectively.

Techniques: Over Expression, Injection, Western Blot, Clinical Proteomics, Control, Staining

Journal: PLoS ONE

Article Title: Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes

doi: 10.1371/journal.pone.0162384

Figure Lengend Snippet: CCF-STTG1 (CCF) astrocytoma cells were treated with DMSO alone, GW3965, or compound 82879 for 48 h. ( A ) A representative Western Blot. Protein levels in whole cell lysates were determined by densitometry for ( B ) apoE ( C ), ABCA1 ( D ), LDLR. Data were normalized over β-actin levels ( A ) and expressed as fold-change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Graphs represent mean and 95% CI from N independent experiments indicated in brackets. Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, *** p< 0.001).

Article Snippet: After blocking with 5% non-fat milk in PBS for 1 h, membranes were probed overnight at 4°C with 1:2000 goat-anti-human apoE (Chemicon, cat# AB947), 1:1000 goat anti-murine apoE (Santa Cruz, cat# sc-6384), 1:2000 monoclonal anti-ABCA1 (a gift from Dr. Michael Hayden [ ]), 1:2000 goat-anti-human LDLR (R&D Systems, cat# AF2148) or 1:5000 anti-β-actin.

Techniques: Western Blot, Blocking Assay