Journal: Nature cell biology

Article Title: Synthetic immunomodulation with a CRISPR super-repressor in vivo

doi: 10.1038/s41556-020-0563-3

Figure Lengend Snippet: (A) Top: Schematic of experiments demonstrating delivery of AAV-Myd88 or Mock repressors to Cas9 nuclease transgenic mice; Bottom: Analysis of anti-AAV1 IgG2A antibody by ELISA. Optical density values are quantified relative to the AAV1/Mock group (N=8 mice). (B) Top: Schematic of experiments demonstrating Cas9 transgenic mice treated with AAV1/Myd88 or AAV1/Mock at day 1, followed by a second administration of AAV1/Mock on day 21; Bottom: Anti-AAV1 IgG1 and total IgG antibody measured by ELISA at different time points. Optical density values are quantified relative to a value of AAV1/Mock+AAV1/Mock group (N=4 mice). (C) Top: Schematic of experiments demonstrating Cas9 transgenic mice treated with AAV1/Myd88 or AAV1/Mock at day 1, followed by a second administration of AAV9/SaCas9 on day 21; Bottom: Analysis of Anti-AAV9 IgG and Anti Sa-Cas9 levels in mice sera. Optical density values are quantified relative to the AAV1/Mock+AAV9/SaCas9 group (N=4 mice). (D) Top: Schematic of experiments demonstrating Cas9 transgenic mice treated with AAV1/Myd88 or AAV1/Mock at day 1, followed by a second administration of AAV9/LacZ on day 21; Bottom: Analysis of Anti-AAV9 IgG in mice sera and LacZ mRNA levels in blood. Relative optical density values are quantified relative to the AAV1/Mock+AAV9/LacZ group (N=4 mice). (E) Top: Schematic of the experiment. Cas9 nuclease transgenic mice were treated with AAV1/Myd88 or AAV1/Mock vectors via retro-orbital injection followed by a second and third injection of AAV9/PCSK9 vectors on day 7 and 21. (F) Analysis of anti-AAV9 IgG and total IgG antibody measured by ELISA. Optical density values are quantified relative to the AAV1/Mock+AAV9/PCSK9+AAV9/PCSK9 group (N=4 mice). (G and H) Plasma samples collected from treated animals were assayed for (G) PCSK9 and (H) Cholesterol at days 0, 7, 21, and 30 (N=4 mice). Panel A-H data are expressed as mean + S.E.M. (Mock= Mock-HP1aKRAB, Myd88= Myd88-HP1aKRAB, PCSK9= PCSK9-HP1aKRAB). Statistical analyses were performed using the non-parametric one-tailed Mann-Whitney U test. A p value ≤ 0.05 was considered significant (*p ≤ 0.05, and ****p ≤ 0.0001). Statistical source data are provided in .

Article Snippet: Plasma Pcsk9 protein levels were quantified by ELISA according to the manufacturer’s instructions (R&D Systems #MPC900).

Techniques: Transgenic Assay, Enzyme-linked Immunosorbent Assay, Injection, Clinical Proteomics, One-tailed Test, MANN-WHITNEY

Journal: Nature cell biology

Article Title: Synthetic immunomodulation with a CRISPR super-repressor in vivo

doi: 10.1038/s41556-020-0563-3

Figure Lengend Snippet: (A) Schematic of experiments demonstrating Cas9 transgenic mice treated with AAV1/Myd88 or AAV1/Mock at day 1, followed by a second administration of AAV1/Mock on day 21. (B) qRT-PCR analysis of Myd88 expression level in lung, blood, and bone marrow of Cas9 transgenic mice (N = 4 mice). Fold changes are relative to universal control. The bars represent the mean + S.E.M. (C) Schematic of the experiment. Cas9 nuclease transgenic mice were treated with AAV1-Myd88 or AAV1-Mock vectors via retro-orbital injection followed by a second and third injection of AAV9-PCSK9 vectors on day 7 and 21. (D) qRT-PCR analysis of Myd88 expression level in lung, blood, and bone marrow of Cas9 transgenic mice (N = 4 mice). The bars represent the mean + S.E.M. (Mock= Mock-HP1aKRAB, Myd88= Myd88-HP1aKRAB, PCSK9= PCSK9-HP1aKRAB). Fold changes are relative to universal control. Universal control is a blood sample collected from an uninjected Cas9 transgenic mouse. Statistical analysis was performed using the non-parametric one-tailed Mann-Whitney U test. A p value ≤ 0.05 was considered significant (*P ≤ 0.05). Statistical source data are provided in .

Article Snippet: Plasma Pcsk9 protein levels were quantified by ELISA according to the manufacturer’s instructions (R&D Systems #MPC900).

Techniques: Transgenic Assay, Quantitative RT-PCR, Expressing, Control, Injection, One-tailed Test, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Full fabrication and application schematic diagram of GelMA-VEGF/ECM-PCSK9 composite hydrogel and the related signaling pathway of PCSK9 that promotes BMSC osteogenic differentiation.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques:

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Construction and characterization of GelMA-VEGF/ECM-PCSK9 composite hydrogel. A Schematic diagram showing the process of composite hydrogel construction; B) Photographs of GelMA-VEGF hydrogel and GelMA-VEGF/ECM-PCSK9 hydrogel formation after UV light respectively; C i) Electron microscopic image of pure GelMA hydrogel, with a scale of 100 μm; ii) Enlarged electron microscopic image of GelMA hydrogel, with a scale of 50 μm; D) i The electron microscope image of the combination of GelMA hydrogel and ECM, with a scale of 100 μm; ii Electron microscope magnified image of GelMA hydrogel combined with ECM, with a scale of 50 μm; E) The infrared spectrum (FITR) diagram of the acellular ECM, GelMA hydrogel and GelMA/ECM composite hydrogel contains common basic energy groups; F) Load rate of PCSK9 in ECM; G) Release rate of VEGF loaded with GelMA hydrogel and GelMA/ECM composite hydrogel respectively; H) Release rate of PCSK9 loaded with ECM and GelMA/ECM composite hydrogel respectively; I) Release rate of VEGF and PCSK9 loaded in GelMA and GelMA/ECM on different time points respectively; J) Release rate of VEGF and PCSK9 respectively when loaded in GelMA/ECM; K) The swelling rate of GelMA gel and GelMA/ECM composite gel dissolved in PBS (n = 6); L) Degradation rate of GelMA hydrogel and GelMA/ECM composite gel in vitro (n = 6).∗means that compared with the control group, p < 0.05; ∗means that compared with the control group, p < 0.01; ∗∗∗means that compared with the control group, p < 0.001.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Microscopy, In Vitro, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Angiogenic capacity formulations of HUVECs in response to different composite biomaterial in vitro. A) Calcein/PI staining of HUVECs seeded on glass slides, showing the cell migration profiles of HUVECs treated with different material groups, scale bar = 200 μm; B) Quantitative analysis of the intercellular blank areas in each group, with the baseline group serving as the negative control; C) Angiogenic images of HUVECs co-cultured with different composite materials for 4 h and 8 h respectively, scale bar = 250 μm; D–G) Quantitative assessment of angiogenic capacity in each group via ImageJ software analysis of key angiogenic parameters. Abbreviations: NC = negative control group; V = exogenous VEGF protein-only group; GV=GelMA + exogenous VEGF protein group; GVE = GelMA + VEGF + ECM group; GVEP= GelMA/VEGF + ECM/PCSK9 group. Statistical notations: ∗∗means that compared with the control group, p < 0.01; ns = no significant difference between group.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Migration, Negative Control, Cell Culture, Software, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: The effect of different composite hydrogel on the osteogenic differentiation of BMMSC in vitro. Cultivate BMMSC for osteogenic differentiation in osteogenic medium with GelMA, GelMA-VEGF, GelMA-VEGF/ECM, ECM-PCSK9, and GelMA-VEGF/ECM-PCSK9 for 7 days respectively. A,B) The cell nucleus was stained with DAPI (blue), RUNX2 was stained with RUNX2 antibody (green), and COL1A1 was stained with COL1A1 antibody (red), with a scale bar of 200 μm. C,D) The quantitative analysis results of COL1A1 and RUNX2 immunofluorescence images; E,F) Quantitative analysis of ALP staining and ARS staining for BMMSC co-culture with different kinds of hydrogels; G) ALP staining result for BMMSC co-culture with different kinds of hydrogels for 7days, scale bar = 200 μm; F) ARS staining result for BMMSC co-culture with different kinds of hydrogels for 14days, scale bar = 200 μm; I, J) After 7 and 14 days of co-culture with different combinations of composite hydrogels and BMMSC for osteogenesis and differentiation, the PCR experiment results of osteogenesis related indicators suggest that compared with the control group. G = simple GelMA hydrogel group, GV=GelMA hydrogels + VEGF protein group, GV/E = GelMA + VEGF/ECM group, EP = ECM + PCSK9 protein group, GVEP=GelMA + VEGF/ECM + PCSK9 protein group, the significant differences between the groups are expressed as ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns means there is no significant difference between the groups.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Immunofluorescence, Co-Culture Assay, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: After adding different concentrations of PCSK9 to BMMSC for osteogenic induction, western blotting (WB) experiment was performed to evaluate the expression of phosphorylated proteins and total proteins among different osteogenic differentiation relevant signaling pathways. A) WB images of different signaling pathways that related to osteogenic differentiation after adding different concentrations of PCSK9; B-D) Quantitative analysis results of phosphorylated protein and total protein. Compared with the control group, ∗ means p < 0.05, ∗∗ means p < 0.01.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Western Blot, Expressing, Protein-Protein interactions, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk

doi: 10.3389/fnagi.2021.632655

Figure Lengend Snippet: Clinical characteristics of study patients by sex.

Article Snippet: PCSK9 levels were measured with commercial enzyme-linked immunosorbent assays (ELISA) kit (#DPC900, R&D) according to the Manufacturer's instructions.

Techniques: Clinical Proteomics, Medications, Battery

Journal: Frontiers in Aging Neuroscience

Article Title: Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk

doi: 10.3389/fnagi.2021.632655

Figure Lengend Snippet: Correlations between circulating PCSK9 and cognitive parameters.

Article Snippet: PCSK9 levels were measured with commercial enzyme-linked immunosorbent assays (ELISA) kit (#DPC900, R&D) according to the Manufacturer's instructions.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk

doi: 10.3389/fnagi.2021.632655

Figure Lengend Snippet: Correlations between circulating PCSK9 levels and short-term memory, as reflected by FDS z-score in female (A) and male (B) patients.

Article Snippet: PCSK9 levels were measured with commercial enzyme-linked immunosorbent assays (ELISA) kit (#DPC900, R&D) according to the Manufacturer's instructions.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk

doi: 10.3389/fnagi.2021.632655

Figure Lengend Snippet: Correlations between waist circumference, circulating PCSK9 levels and short-term memory, as reflected by FDS z-score in female (A,C) and in male (B,D) .

Article Snippet: PCSK9 levels were measured with commercial enzyme-linked immunosorbent assays (ELISA) kit (#DPC900, R&D) according to the Manufacturer's instructions.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk

doi: 10.3389/fnagi.2021.632655

Figure Lengend Snippet: Sex-specific, multivariable association of cognitive performance in the memory domain with PCSK9 in subjects at high cardiovascular risk.

Article Snippet: PCSK9 levels were measured with commercial enzyme-linked immunosorbent assays (ELISA) kit (#DPC900, R&D) according to the Manufacturer's instructions.

Techniques:

Journal: Scientific Reports

Article Title: ITCH modulates SIRT6 and SREBP2 to influence lipid metabolism and atherosclerosis in ApoE null mice

doi: 10.1038/srep09023

Figure Lengend Snippet: (a) mRNA expression analysis for genes involved in cholesterol metabolism in the liver (N = 4). (b) Representative western blots for LDL receptor and PCSK9 from livers of ApoE−/− and ApoE−/−ITCH−/− (N = 6 per group). (c) PCSK9 serum levels from ApoE-/- and ApoE-/-ITCH-/- mice (N = 6 per group) (d) Representative western blot and densitometry of nuclear extracts from livers of ApoE−/− and ApoE−/−ITCH−/− mice. (e) Effect of ITCH knockdown by increasing concentrations of SiRNA in HepG2 cells on ITCH and SREBP2 (cytisolic, c; nuclear, n) protein levels. Data are presented as Mean +/− SEM. ***P < 0.001, **P < 0.01 and *P < 0.05 versus ApoE −/− .

Article Snippet: Western blotting and immunoprecipitation were performed with the following antibodies: SIRT1 (ab28170), SIRT6 (ab62739), SREBP2 (ab30682), LDL receptor (ab52818), ITCH (BD pharmigen 611198), PCSK9 (R&D biosystems AF3985).

Techniques: Expressing, Western Blot, Knockdown

Journal: iScience

Article Title: Macrophage AMPK β1 activation by PF-06409577 reduces the inflammatory response, cholesterol synthesis, and atherosclerosis in mice.

doi: 10.1016/j.isci.2023.108269

Figure Lengend Snippet: Figure 2. PF-06409577 reduces atherosclerosis via macrophage AMPK activation (A–D) Peritoneal macrophages were isolated from AMPK b1fl/fland AMPK b1LysM mice. (A) AMPK levels and phosphorylation of ACC following treatment with PF- 06409577 (10 mM, 90 min) were detected by western blot and (B–D) quantified using ImageJ data are presented as mean G SEM, * indicates p < 0.05 and *** indicates <0.005 by unpaired t test. AMPK b1fl/fland AMPK b1 LysM mice were injected with PCSK9 AAV, fed a Western diet and treated with Vehicle or 100 mg/kg PF-06409577 by oral gavage for 6 weeks. (E and F) Representative plaque images, scale bar represents 100 mM and quantification. Data are presented as mean G SEM, # indicates p < 0.05 in overall group effect my two-way ANOVA, * indicates p < 0.05 by two-way ANOVA with Fisher’s LSD post-hoc testing.

Article Snippet: Serum PCSK9 levels were analysed using a murine PCSK9 ELISA (Cat# MPC900, R&D Systems) as per the manufacturer’s instructions.

Techniques: Activation Assay, Isolation, Phospho-proteomics, Western Blot, Injection

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 1. Schematic representation of PCSK9 constructs used in this study. PCSK9 comprises a signal peptide (SP), a prodomain (Pro), a catalytic domain, a HR, and a CHRD. The numbers and arrows denote amino acid positions at boundaries between domains. The N-terminal CAT construct and the HR-CHRD were individually subcloned for further study. Full-length PCSK9 and the truncated constructs (CAT and HR-CHRD) were fused with the TM-CT of LAMP1 or ACE2 to drive the proteins to the lysosomal compartment or to the cell surface, respectively.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Construct

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 2. Recruitment of PCSK9 to the cell surface by LDLR and CD81. A–C, immunofluorescence analysis of PCSK9 localization. In addition to peGFP as an indicator of transfection, Huh7 cells were co-transfected with plasmids expressing PCSK9 and LDLR (A, upper), PCSK9 and CD81 (A, lower), PCSK9 (B), or empty vector (C). Cells were not permeabilized and were stained with rabbit anti-PCSK9 (red) and goat-anti LDLR (A, upper, green) or rabbit anti-PCSK9 and mouse anti-CD81 (B, lower, green) antibodies. D--I, verification of antibody specificity. Huh7 cells were co-transfected with peGFP and PCSK9 (D), CD81 (F), LDLR (H), or empty vector (E, G, and I). Transfected cells were treated with Triton X-100 (D and E) or left untreated (F–I) and stained with rabbit anti-PCSK9 (D and E), rabbit anti-PCSK9 and mouse anti-CD81 (F and G), or rabbit anti-PCSK9 and goat anti-LDLR (H and I) antibodies. Following primary antibody incubation, cells were stained with Alexa Fluor 568 chicken anti-rabbit IgG (D--I, red). The nuclei were stained with DAPI. The co-localization measurements in A were based on Manders’ coefficients using ImageJ and JACoP.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Staining, Incubation

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 3. Effects of PCSK9 constructs on endogenous LDLR and CD81 expression in Huh7 cells. A, immunofluorescence analysis of PCSK9 construct localization. Huh7 cells were co-transfected with plasmids expressing eGFP and wild-type PCSK9 (left panel) or eGFP and PCSK9 fused to the TM-CT of ACE2 (rightpanel).Anti-PCSK9staining(red)wascomparedinnon-permeabilized(()Triton)andpermeabilized(()Triton)Huh7cells.B,Westernblotanalysisofthe expression and maturation of overexpressed PCSK9 constructs in Huh7 cells using rabbit anti-PCSK9. Cells were transfected with plasmids encoding the indicated proteins. C, effects of PCSK9-ACE2 on total and surface expressions of LDLR and CD81 in Huh7 cells. The cells were transfected with plasmids encoding PCKS9-ACE2, ACE2, or an empty vector and stained with anti-LDLR or anti-CD81 antibodies prior to analysis by FACS. Saponin was added when the detection of intracellular protein was required (total LDLR and total CD81). The experiments were repeated at least three times. Statistics analysis was based on one-way analysis of variance (ANOVA) Dunnett’s test. *, p 0.05; ***, p 0.001. The error bars represent S.E.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Construct, Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Staining

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 4. Effects of PCSK9 mutations in the signal peptide or prodo- main on endogenous LDLR and CD81 expression. Huh7 cells were transfected with plasmids coding for PCSK9-ACE2 mutants in the signal peptide or prodomain. A, Western blot analysis of PCSK9-ACE2 mutant expression and maturation using rabbit anti-PCSK9 antibody. B, FACS analysis of surface expression of the LDLR and CD81 in Huh7 cells trans- fected with PCSK9-ACE2 mutants. All experiments were repeated at least three times. Statistics analysis was based on one-way ANOVA Dunnett’s test. *, p 0.05; **, p 0.01; ***, p 0.001. The error bars represent S.E. M/P, mature PCSK9 to pro-PCSK9.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, Western Blot, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 5. Effects of PCSK9 mutations in the CAT on endogenous LDLR and CD81 expression. Huh7 cells were transfected with plasmids coding for PCSK9-ACE2 mutants in the CAT. A, Western blot analysis of PCSK9 expression and maturation using rabbit anti-PCSK9 antibody. B, FACS analysis of surface expression of the LDLR and CD81 in Huh7 cells transfected with PCSK9-ACE2 mutants. All experiments were repeated at least three times. Statistics analy- sis was based on one-way ANOVA Dunnett’s test. *, p 0.05; **, p 0.01; ***, p 0.001. The error bars represent S.E. M/P, mature PCSK9 to pro-PCSK9; NA, not applicable.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 6. Effects of PCSK9 mutants in HR-CHRD on endogenous LDLR and CD81 expression. Huh7 cells were transfected with plasmids coding for PCSK9-ACE2 mutants in the HR-CHRD. A, Western blot analysis of PCSK9 expression and maturation using rabbit anti-PCSK9 antibody. B, FACS analysis of surface expression of the LDLR and CD81 in Huh7 cells transfected with PCSK9 mutants. All experiments were repeated at least three times. Statistics analysis was based on one-way ANOVA Dunnett’s test. *, p 0.05; **, p 0.01; ***, p 0.001. The error bars represent S.E. M/P, mature PCSK9 to pro-PCSK9.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 7. Effects of CAT and HR-CHRD on the endogenous LDLR and CD81 expression. Huh7 cells were transfected with plasmids coding for PCSK9 constructs (full length, CAT, and HR-CHRD) with or without the TM-CT of ACE2. A, Western blot analysis of PCSK9 expression and matura- tion using rabbit anti-PCSK9 antibody. B, FACS analysis of surface expres- sion of the LDLR and CD81 in Huh7 cells transfected with PCSK9 con- structs. All experiments were repeated at least three times. Statistic analysis was based on one-way ANOVA Dunnett’s test. **, p 0.01; ***, p 0.001. The error bars represent S.E.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, Construct, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 8. Effects of intracellular PCSK9 on total and surface endogenous LDLR and CD81 expression. A, Huh7 cells were transfected with plasmids expressing wild-type PCSK9 or PCSK9 with the TM-CT of LAMP1 protein (PCSK9-LAMP1) that targets the protein to the lysosomes. Total cellular proteins were separated by 10% SDS-PAGE and then analyzed by rabbit anti-PCSK9 antibody. B, localization of PCSK9-LAMP1 in the cells was analyzed by immunofluorescence. Huh7 cells were co-transfected with eGFP- and PCSK9-LAMP1-coding plasmids before staining with rabbit anti-PCSK9 antibody. To detect intracellular PCSK9, cells were treated with Triton X-100. C, co-localization of PCSK9-LAMP1 with a lysosome marker. Huh7 cells were transiently transfected with PCSK9-LAMP1. Before fixation and staining with rabbit anti-PCSK9 (green), cells were incubated with LysoTracker (red). Imaging was done by a confocal microscope. The co-localization measurement was based on Manders’ coefficients using ImageJ and JACoP. D, effects of PCSK9-LAMP1 on surface and total expressions of LDLR and CD81 in Huh7 cells by FACS. The cells were transfected with PCSK9-LAMP1, LAMP1, or empty vector as a control and then subjected to FACS using anti-LDLR or anti-CD81 antibody. Intracellular detection was obtained through saponin treatment (total LDLR and total CD81). The experiments were repeated at least three times. Statistics analysis was based on one-way ANOVA Dunnett’s test. *, p 0.05; ***, p 0.001. The error bars represent S.E.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, SDS Page, Immunofluorescence, Staining, Marker, Incubation, Imaging, Microscopy, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 9. Interaction among PCSK9 and endogenous membrane-anchored proteins LDLR and CD81 by in situ PLA. Huh7 cells were co-transfected with eGFP-andPCSK9-ACE2-encodingplasmidsoreGFPandemptyphCMV3vectorasacontrolandgrownoncoverslips.Thecellswerepermeabilized,andthePLA procedure was performed according to the manufacturer’s instructions using the rabbit anti-PCSK9 and mouse anti-LDLR or rabbit anti-PCSK9 and mouse anti-CD81antibodies.DuolinkImageToolsoftwarewasusedtoanalyzePLAsignals(reddots).Histograms(right)showquantificationofspecificandnonspecific signal. Statistics analysis was based on unpaired t test. ***, p 0.001. The error bars represent S.E.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Membrane, In Situ, Transfection

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE 10. CoIP of CD81 with PCSK9. HEK293T cells were co-transfected with CD81- and PCSK9-V5- (A), PCSK9-ACE2- (B, upper), CD81- and ACE2- (B, lower),orPCSK9-V5-andLDLR(C)-encodingplasmids.Celllysateswerepulled down using anti-CD81 (clone 5A6) (A and B) or anti-V5 (C) antibody; mouse IgG1 was used as the mock antibody. A and B, coIP of CD81 with PCSK9 (A) or PCSK9-ACE2 but not ACE2 (B) using anti-CD81. C, coIP of PCSK9-V5 with LDLR using anti-V5. The red asterisk pinpoints PCSK9, which partially overlaps with the mouse 5A6 heavy chain. CE, cells extract.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Transfection

Journal: Journal of Biological Chemistry

Article Title: Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

doi: 10.1074/jbc.m115.642991

Figure Lengend Snippet: FIGURE11.RecruitmentofPCSK9byCD81anditsfunctioninmodulationofLDLRandCD81inLDLRnon-expressingCHOcells.A,parentalCHOcellsand CHO cells lacking LDLR expression (CHO-A7) were co-transfected with a set of plasmids expressing eGFP, PCSK9, and CD81 or eGFP, PCSK9, and empty phCMV3 vector as a negative control. Cells were stained with rabbit anti-PCSK9 (red) and mouse-anti CD81 (green) antibodies. The nuclei were stained with DAPI. Imaging was done by a confocal microscope. B, effect of PCSK9 on LDLR in CHO-A7 cells. In addition to eGFP- and LDLR-encoding plasmids, CHO and CHO-A7 cellswereco-transfectedwithACE2(red),PCSK9-ACE2(blue),oremptyvector(gray).Cellswerestainedwithallophycocyanin-conjugatedmouseanti-LDLRand subjected to FACS analysis. The histograms represent triplicate experiments. C, in addition to eGFP-encoding plasmid as the indicator of transfection, CHO and CHO-A7 cells were co-transfected with CD81 and plasmids coding for the indicated proteins. Cells were stained with phycoerythrin-conjugated mouse anti-CD81 and subjected to FACS analysis. Expressions of CD81 in transfected CHO cells were compared with empty vector-transfected cells. The experiments were repeated at least three times. Statistics analysis was based on one-way ANOVA Tukey test. *, p 0.05; **, p 0.01; ***, p 0.001; ns, not significant. The error bars represent S.E.

Article Snippet: The membranes were blocked for 1 h and then incubated with in-house rabbit anti-human PCSK9 antibody (1:2500), goat anti-ACE2 antibody (clone C-18, 1:1000; Santa Cruz Biotechnology), or mouse anti- -actin antibody (1:1000; Ambion) for 1 h. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories) for 1 h. The membranes were incubated with enhanced chemiluminescent HRP substrate and exposed to x-ray films.

Techniques: Expressing, Transfection, Plasmid Preparation, Negative Control, Staining, Imaging, Microscopy

Journal: European journal of clinical investigation

Article Title: PCSK9 and coronary atherosclerosis progression beyond LDL-cholesterol in coronary artery disease patients.

doi: 10.1111/eci.70083

Figure Lengend Snippet: FIGURE 1 Association between circulating levels of plasma PCSK9 with lipid (Total-C, LDL-C and HDL-C) and inflammatory (IL6, MMP9 and ICAM1) profiles of study patients.

Article Snippet: HUVEC were stimulated with .25, 1, 2.5 or 5 μg/mL human PCSK9 Protein (ACRObiosystem, Newark, DE, USA) for 4 h to evaluate mRNA expression of VCAM- 1, ICAM- 1, MCP- 1, IL- 6, IL- 8 and for 24 h to assess the protein surface exposure of VCAM- 1 and ICAM- 1. nloaded from https://onlinelibrary.w iley.com /doi/10.1111/eci.70083 by IN A SP - N E PA L , W iley O nline L ibrary on [07/06/2025].

Techniques: Clinical Proteomics

Journal: European journal of clinical investigation

Article Title: PCSK9 and coronary atherosclerosis progression beyond LDL-cholesterol in coronary artery disease patients.

doi: 10.1111/eci.70083

Figure Lengend Snippet: FIGURE 2 Association of PCSK9 plasma levels and plaque progression. In univariate and multivariate models, data are depicted as β coefficients with 95% CIs for the annual change in plaque volume from baseline to follow-up coronary CTA.

Article Snippet: HUVEC were stimulated with .25, 1, 2.5 or 5 μg/mL human PCSK9 Protein (ACRObiosystem, Newark, DE, USA) for 4 h to evaluate mRNA expression of VCAM- 1, ICAM- 1, MCP- 1, IL- 6, IL- 8 and for 24 h to assess the protein surface exposure of VCAM- 1 and ICAM- 1. nloaded from https://onlinelibrary.w iley.com /doi/10.1111/eci.70083 by IN A SP - N E PA L , W iley O nline L ibrary on [07/06/2025].

Techniques: Clinical Proteomics

Journal: European journal of clinical investigation

Article Title: PCSK9 and coronary atherosclerosis progression beyond LDL-cholesterol in coronary artery disease patients.

doi: 10.1111/eci.70083

Figure Lengend Snippet: FIGURE 3 RNA-sequencing results (A) Molecular pathway enrichment by STRING linking circulating PCSK9-related gene dataset with KEGG databases; (B) PCSK9 and MMP9 plasma levels together with annual change of Necrotic Core and Fibrous PVs divided according to presence of PCSK9 gene expression in whole blood; (C) Functional annotation of PCSK9-related genes according to GO Biological Processes. The 20 most significant biological processes are shown. The GeneRatio indicates how many PCSK9-related genes included in the analysis were annotated to the specific GO biological process. GO terms were filtered for adjusted p-value <.01; (D) Visual combination of genes with related biological processes for enhanced graphical representation of functional categories related to innate immunity.

Article Snippet: HUVEC were stimulated with .25, 1, 2.5 or 5 μg/mL human PCSK9 Protein (ACRObiosystem, Newark, DE, USA) for 4 h to evaluate mRNA expression of VCAM- 1, ICAM- 1, MCP- 1, IL- 6, IL- 8 and for 24 h to assess the protein surface exposure of VCAM- 1 and ICAM- 1. nloaded from https://onlinelibrary.w iley.com /doi/10.1111/eci.70083 by IN A SP - N E PA L , W iley O nline L ibrary on [07/06/2025].

Techniques: RNA Sequencing, Clinical Proteomics, Gene Expression, Functional Assay

Journal: European journal of clinical investigation

Article Title: PCSK9 and coronary atherosclerosis progression beyond LDL-cholesterol in coronary artery disease patients.

doi: 10.1111/eci.70083

Figure Lengend Snippet: FIGURE 4 In vitro study. (A) HUVECs were treated with PCSK9 (.25–5 μg/mL) for 4 h and RT-PCR was performed with specific primers for VCAM-1, ICAM-1, and RPL13a; (B) ICAM-1 and VCAM-1 surface exposure in HUVEC treated with PCSK9 (.25–5 μg/mL) overnight. At the end of the incubation time, VCAM-1 and ICAM-1 surface exposure was quantified by EIA. Values are mean ± SD of optical density arbitrary units (AU) at 405 nm; (C) HUVECs were treated with PCSK9 (.25–5 μg/mL) for 4 h and RT-PCR was performed with specific primers for MCP-1, IL6, IL8 and RPL13a.

Article Snippet: HUVEC were stimulated with .25, 1, 2.5 or 5 μg/mL human PCSK9 Protein (ACRObiosystem, Newark, DE, USA) for 4 h to evaluate mRNA expression of VCAM- 1, ICAM- 1, MCP- 1, IL- 6, IL- 8 and for 24 h to assess the protein surface exposure of VCAM- 1 and ICAM- 1. nloaded from https://onlinelibrary.w iley.com /doi/10.1111/eci.70083 by IN A SP - N E PA L , W iley O nline L ibrary on [07/06/2025].

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Incubation