Journal: Cell Reports Medicine

Article Title: Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia

doi: 10.1016/j.xcrm.2024.101614

Figure Lengend Snippet:

Article Snippet: Recombinant Anti-LDLR/LDL Receptor Antibody (APC), Rabbit Monoclonal , SinoBiological , Cat#10231-R301-A, RRID: AB_3086609.

Techniques: Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Staining, Cell Stimulation, Adjuvant, Western Blot, Reverse Transcription, Plasmid Preparation, Protein Extraction, RNA Extraction, Software

Journal: Cell

Article Title: Oxysterol restraint of cholesterol synthesis prevents AIM2 inflammasome activation

doi: 10.1016/j.cell.2017.09.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse LDLR Polyclonal Antibody, PE , Sino Biological , Cat# 50305-R032-P-25.

Techniques: Recombinant, Isolation, Amplex Red Cholesterol Assay, Plasmid Preparation, Software

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Effects of ribosomal inactivation on intracellular fat and gene profiles. (A-D) HCT-8 (A and B) and HepG2 (C and D) cells were treated with the vehicle, 1000 ng/mL RIS-1, or 2 mM RIS-2 for 24 h. (A and B) Lipid droplets were detected using a transmission electron microscope (TEM) at the original magnification x1000 (scale bar(s), 2 mm) with white arrows indicating lipid droplets in each cell. (C and D) Total lipids from the whole cell lysate were analyzed for lipidomic profiling using mass spectrometry (performed by Lipotype GmbH, Dresden, Germany). (E) Pearson 's correlation analysis of relationships between transcription levels of key components of the ribotoxic stress responses and lipid metabolism in patients with IBD (gse117993 ( Denson 's, n = 190)). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). (F) HCT-8 cells were treated with the vehicle or 500 ng/mL RIS-1 for 1 h. Functional gene ontology using RNA sequencing analysis of the cellular mRNA was evaluated based on p values for each category. (G-H) HCT-8 (G) and HepG2 (H) cells were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 24 h. Each mRNA was measured using reverse transcription-quantitative PCR. (I-K) LDL uptake into human intestinal epithelial cells (upper panels) and cellular LDLR expression (lower pannels) were measured using LDL-Dylight TM 550 (red) and DyLight TM -488-conjugated anti-LDLR antibody (green), respectively. The microscopic analysis was performed at the original magnification of 200× (scale bar(s), 50 μm) with each quantitative graph (J and K, * p < 0.05, ** p < 0.01, and *** p < 0.001). LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; CE, cholesterol esters; DAG, diacylglycerol; PA, phosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine (-ether); PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TAG, triacylglycerol.

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Transmission Assay, Microscopy, Mass Spectrometry, Generated, Expressing, Functional Assay, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: The ribotoxic stress responses modulate intestinal LDLR expression. (A-B) Mice were treated with the vehicle or 25 mg/kg RIS-1 for 24 h via oral gavage. Ileal and jejunal tissue specimens were subjected to immunohistochemistry to detect LDLR expression (A). The microscopic analysis was performed at the original magnification of 100× (scale bar(s), 100 μm), followed by quantitative analysis using HistoQuest software (B). (C) LDLR expression was assessed in patients with IBD (gse75214 ( Vemeire 's, n = 194) and gse117993 ( Denson 's, n = 190)). Based on EIF2AK2 levels, we selected samples exhibiting the 50 highest and 50 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (* p < 0.05, ** p < 0.01, *** p < 0.001). (D) Pearson 's correlation analysis of relationships between transcription levels of critical components of the ribotoxic stress responses and cell-type-specific markers in patients with IBD (gse117993 ( Denson 's, n = 190)). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). IBD, inflammatory bowel disease; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor. (E-G) Analysis based on scRNA-Seq dataset from patients with CD (gse202052 (E) and gse156776 (F)). (E-F) UMAP clusters for gse202052 (E) and gse156776 (F). (G) Based on EIF2AK2 or ZAK levels, we selected samples exhibiting the 500-1500 highest and 500-1500 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (**** p < 0.0001). (H-I) Analysis based on scRNA-Seq dataset from mouse intestine fed with HFD for 7 days (gse199776). (H) UMAP clusters. (I) Based on EIF2AK2 or ZAK levels, we selected samples exhibiting the 500 highest and 500 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (** p < 0.01, *** p < 0.001).

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemistry, Software, Whisker Assay, Generated

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: EIF2AK2 mediates LDLR expression in the ribosome-insulted gut. (A-D) Small intestine-derived organoids from the wild-type mice were treated with vehicle, RIS-1 (500 ng/mL), RIS-2 (1 μM), or EIF2AK2 inhibitor (0.5 μM, EIF2AK2i) for 24 h. (A) Representative Oil Red O staining for the intestinal organoids. The microscopy analysis was performed at the original magnification of 100×; scale bar(s), 100 μm. (B) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in the gut organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (C) The confocal microscopic analysis of LDLR expression was performed at the original magnification of 200×; scale bar(s), 50 μm. (D) The graph shows the relative quantitative values for LDLR protein in the intestinal organoids using ImageJ software. Different letters over each bar represent significant differences between groups ( p < 0.05). (E-G) HCT-8 cells transfected with the negative control vector or shEIF2AK2 were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2. (E) Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope. The microscopy analysis was performed at the original magnification of 200×; scale bar(s), 50 μm. (F) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in cells using ImageJ software (*** p < 0.001). (G) HCT-8 cells expressing the negative control or EGR1-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR (*** p < 0.001).

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Derivative Assay, Staining, Microscopy, Software, Transfection, Negative Control, Plasmid Preparation, Light Microscopy, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: LDLR mediates fat deposition in the gut (A) Ten-week-old male C57BL/6J wild-type or LDLR knockout (KO) mice were treated with the vehicle or 25 mg/kg RIS-1 for 24 h via oral gavage. Representative Oil Red O staining for the small intestine. (B) Relative quantitative values of Oil Red O-positive lipid droplets in small intestines using ImageJ software. The microscopy analysis was performed at the original magnification of 400×; scale bar(s), 50 μm. (C) Measurement of serum LDL cholesterol. Results are shown as box-and-whisker plots (Tukey), and different letters over each box represent significant differences between groups ( p < 0.05 using one-way ANOVA with the Newman-Keuls post hoc test). (D-E) HCT-8 cells transfected with the negative control vector or shLDLR were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2. Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope (D). The microscopic analysis was performed at the original magnification of 200×; scale bar(s), 10 μm (left panel). (E) The graph shows relative quantitative values of Oil Red O-positive lipid droplets in cells using ImageJ software (E). The right boxed graph represents the suppression of LDLR mRNA expression by shLDLR (*** p < 0.001). (F) Small intestine-derived organoids from wild-type or LDLR-KO mice were treated with vehicle, RIS-1 (500 ng/mL), or RIS-2 (1 μM) for 24 h. Representative Oil Red O staining for small intestine organoids. The microscopy analysis was performed at the original magnification of 100×; scale bar(s), 100 μm. Different letters over each bar represent significant differences between groups ( p < 0.05). (G) Relative quantitative values of Oil Red O-positive lipid droplets in organoids using ImageJ software. Results are shown as box-and-whisker plots (Tukey), and different letters over each box represent significant differences between groups ( p < 0.05 using one-way ANOVA with the Newman-Keuls post hoc test). LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor.

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Knock-Out, Staining, Software, Microscopy, Whisker Assay, Transfection, Negative Control, Plasmid Preparation, Light Microscopy, Expressing, Derivative Assay

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: LDLR expression in response to ribosomal inactivation is dependent on EGR1 and SREBP2. (A) HCT-8 cells were treated with 1000 ng/mL RIS-1 for the indicated time. The graph shows the quantification of nuclear SREBP2 protein from the confocal microscopic analysis (boxed panels) and LDLR mRNA using reverse transcription-quantitative PCR (RT-qPCR). (B) HCT-8 cells were pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h and then treated with vehicle or 1000 ng/mL RIS-1 for 30 min. The bar graph shows the quantification of nuclear SREBP2 protein using a confocal microscope (** p < 0.01 and *** p < 0.001). (C and D) For the ChIP assay, HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. (E) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time and examined under a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right panel shows the relative quantitative values for nuclear EGR1 protein in cells. The relative ratio was obtained by measuring the nuclear density of EGR1 corresponding to the DAPI-stained area (the right graph, * p < 0.05). (F) HCT-8 cells were treated with vehicle or 1000 ng/mL RIS-1 for the indicated time. Nuclear fractions of cell lysates were subjected to western blot analysis. (G) HCT-8 cells pretreated with the vehicle, 10 μM SP600125 (SP), 10 μM SB203580 (SB), or 2 μM U0126 for 2 h were exposed to vehicle or 1000 ng/mL RIS-1 for 1 h. Nuclear fractions of cell lysates were subjected to western blot analysis. ChIP, chromatin immunoprecipitation; EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Microscopy, Staining, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Transcriptional regulation of LDLR expression in response to ribosomal inactivation. (A) A putative scheme for ribosomal stress-induced transcriptional activation of LDLR expression via SREBP2 activation and EGR1 induction. (B) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. LDLR mRNA was measured using reverse transcription-quantitative PCR. Figures in the box represent the inhibition of EGR-1 and SREBP2 mRNA by each shRNA (*** p < 0.001). (C) HCT-8 cells transfected with the wild-type (wt) or the mutants (SRE mt and CRE mt) of LDLR promoter-containing plasmids were treated with vehicle or 1000 ng/mL RIS-1 for 6 h, and the cellular luciferase activity was measured (* p < 0.05). (D) EGR1- or SREBP2-deficient cells were transiently transfected with wt LDLR promoter-containing plasmid and then treated with vehicle or 1000 ng/mL RIS-1 for 6 h to measure the cellular LDLR promoter activity. Different letters over each bar represent significant differences between the two groups ( p < 0.05). The right boxed graphs show suppression of mRNA expression by each shRNA (*** p < 0.001). (E-F) HCT-8 cells expressing the negative control vector, EGR1-, or SREBP2-specific shRNA were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. Intracellular lipid droplets were stained with Oil Red O and visualized using a light microscope at the original magnification of 200×; scale bar(s), 50 μm (E). The right graph shows the relative quantitative values of Oil Red O-positive lipid droplets, and different letters over each bar represent significant differences between the two groups (F, p < 0.05 using one-way ANOVA with the Newman-Keuls post hoc test). EGR1, early growth response 1; LDLR, low-density lipoprotein receptor; SREBP2, sterol regulatory element-binding protein 2.

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Activation Assay, Negative Control, Plasmid Preparation, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Inhibition, Transfection, Luciferase, Activity Assay, Staining, Light Microscopy, Binding Assay

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: Effects of ribosomal inactivation on LDLR mRNA stability in human intestinal cells. (A) HCT-8 cells were treated with the vehicle or 1000 ng/mL RIS-1 for 2 h and then replaced with 5 μM actinomycin D for the indicated time to arrest cellular transcription. Expression levels of LDLR mRNA were measured using reverse transcription-quantitative PCR (RT-qPCR). The underlying boxed blots represent the mRNA measured by conventional RT-PCR. (B) HCT-8 cells were treated with vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h and examined using a confocal microscope. The microscopic analysis was performed at the original magnification of 1600×; scale bar(s), 20 μm. The right graph shows the relative quantitative values of cytosolic HuR in cells using ImageJ software (*** p < 0.001). (C) HCT-8 cells transfected with the negative control vector or HuR shRNA expression vector (shHuR) were treated with the vehicle, 1000 ng/mL RIS-1, or 2 μM RIS-2 for 2 h. mRNA levels were measured using RT-qPCR. The boxed images show the suppression of HuR mRNA expression by shHuR (*** p < 0.001). (D) A putative scheme for ribosomal inactivation-induced cholesterol uptake. (E) Pearson 's correlation analysis of relationships between transcription levels of critical components of ribosotoxic stress responses and LDLR in the small intestine of diet-induced obese mice (gse199776). Correlation matrix visualization was generated using the corrplot function of the R-package (R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/ ). Correlations of transcriptional expression among genes were interpreted according to a general guideline for Pearson's coefficient value: r > 0.7, high (+); 0.5 < r < 0.7, moderate (+); 0.3 < r < 0.5, moderate (+) or low (+); 0.1 < r < 0.3, low (+); -0.1 < r < 0.1, negligible; -0.3 < r < -0.1, low (-). (F) LDLR expression was assessed in patients with IBD (gse75214; Vemeire 's, n = 194). Based on EGR1, SREBP , or HuR levels, we selected samples exhibiting the 50 highest and 50 lowest levels, which were further compared for LDLR levels. Results are shown as a box-and-whisker plot (Tukey), and asterisks (*) indicate significant differences from the low expression group (* p < 0.05, *** p < 0.001). EGR1, early growth response 1; HuR, human antigen R; IBD, inflammatory bowel disease; LDL, low-density lipoprotein; LDL, low-density lipoprotein receptor; SREBP, sterol regulatory element-binding protein.

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Microscopy, Software, Transfection, Negative Control, Plasmid Preparation, shRNA, Generated, Whisker Assay, Binding Assay

Journal: Theranostics

Article Title: Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction

doi: 10.7150/thno.88586

Figure Lengend Snippet: The forward- and reverse-complement PCR primers for amplification of each gene

Article Snippet: The membranes were then incubated with the following antibodies: rabbit polyclonal anti-human actin antibody (1:1000), rabbit polyclonal anti-human SREPB2 antibody (1:1000), rabbit polyclonal anti-human EGR1 antibody (1:1000), mouse monoclonal anti-human HuR antibody (1:2000), mouse monoclonal anti-human hnRNP antibody (1:2000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human LDLR antibody (1:1000) (Cayman Chemical, Ann Arbor, Michigan, USA) for 2 h at room temperature.

Techniques: Amplification, Sequencing