Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Sequences of custom-made siRNAs directed toward human targets

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Primer sequences for real-time PCR

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: (Pro)renin receptor mediates albumin-induced cellular responses: role of site-1 protease-derived soluble (pro)renin receptor in renal epithelial cells

doi: 10.1152/ajpcell.00006.2017

Figure Lengend Snippet: Effects of siRNA-mediated silencing of PCSK7, PCSK9, and S1P on PRR cleavage induced by BSA in HK-2 cells. The cells were transfected with siRNA against PCSK7, PCSK9, or S1P for 48 h and then treated with 20 mg/ml BSA for 24 h. A and B: immunoblotting of fPRR and sPRR. Shown are representative blots and quantitative densitometry analysis. C and D: ELISA detection of medium sPRR. The values are normalized by protein content. *P < 0.05 vs. CTR; #P < 0.05 vs. BSA; NS, nonsignificance vs. BSA. Data are means ± SD; n = 6/group. C and D values are presented as the median (central line), interquartile range (box), and range (whiskers).

Article Snippet: The knockdown of endogenous furin (catalog no. SR303336), ADAM19 (catalog no. SR305748), proprotein convertase (PC) subtilisin/kexin type 7 (PCSK7; catalog no. SR306062), S1P (catalog no. SR305740), PCSK9 (catalog no. SR316837), or PRR (catalog no. SR306857) was performed using the predesigned small interfering RNA (siRNA) from OriGene (Rockville, MD).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Preventive Cardiology

Article Title: Circulating Endothelial Progenitor Cells in Patients with Established Cardiovascular Disease Treated with PCSK9 Monoclonal Antibodies ☆

doi: 10.1016/j.ajpc.2024.100896

Figure Lengend Snippet: Baseline characteristics of patients before the initiation of PCSK9 mAb a .

Article Snippet: Levels of PCSK9 were measured using ELISA assays according to the manufacturer's recommendations (Human PCSK9 catalog number: DPC900 SPC900 PDPC900, R&D systems).

Techniques: Medications

Journal: American Journal of Preventive Cardiology

Article Title: Circulating Endothelial Progenitor Cells in Patients with Established Cardiovascular Disease Treated with PCSK9 Monoclonal Antibodies ☆

doi: 10.1016/j.ajpc.2024.100896

Figure Lengend Snippet: Lipids profile of study patients before and after initiation of PCSK9 mAb a .

Article Snippet: Levels of PCSK9 were measured using ELISA assays according to the manufacturer's recommendations (Human PCSK9 catalog number: DPC900 SPC900 PDPC900, R&D systems).

Techniques:

Journal: American Journal of Preventive Cardiology

Article Title: Circulating Endothelial Progenitor Cells in Patients with Established Cardiovascular Disease Treated with PCSK9 Monoclonal Antibodies ☆

doi: 10.1016/j.ajpc.2024.100896

Figure Lengend Snippet: cEPCs levels before and following the initiation of PCSK9 mAb. Percentage levels of (A) CD34 (+) /VEGFR-2 (+) and (B) CD133 (+) /VEGFR-2 (+) cells at 1 month and 3 months following the initiation of PCSK9 mAb.

Article Snippet: Levels of PCSK9 were measured using ELISA assays according to the manufacturer's recommendations (Human PCSK9 catalog number: DPC900 SPC900 PDPC900, R&D systems).

Techniques:

Journal: American Journal of Preventive Cardiology

Article Title: Circulating Endothelial Progenitor Cells in Patients with Established Cardiovascular Disease Treated with PCSK9 Monoclonal Antibodies ☆

doi: 10.1016/j.ajpc.2024.100896

Figure Lengend Snippet: CFUs and MTT assay levels before and following the initiation of PCSK9 mAb. Number of EPCs-CFUs (left) and MTT assay (right) performed before and after 1-month and 3-months of treatment with PCSK9 mAb.

Article Snippet: Levels of PCSK9 were measured using ELISA assays according to the manufacturer's recommendations (Human PCSK9 catalog number: DPC900 SPC900 PDPC900, R&D systems).

Techniques: MTT Assay

Journal: American Journal of Preventive Cardiology

Article Title: Circulating Endothelial Progenitor Cells in Patients with Established Cardiovascular Disease Treated with PCSK9 Monoclonal Antibodies ☆

doi: 10.1016/j.ajpc.2024.100896

Figure Lengend Snippet: cEPCs levels and function, at time 0, 1 month and 3 months following the initiation of PCSK9 mAb.

Article Snippet: Levels of PCSK9 were measured using ELISA assays according to the manufacturer's recommendations (Human PCSK9 catalog number: DPC900 SPC900 PDPC900, R&D systems).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: PCSK9 is expressed and secreted by pancreatic beta cells . A – C , PCSK9 mRNA expression in ( A ) mouse liver compared with FACS-enriched pancreatic beta, delta, and alpha endocrine fractions (n = 4–5); ( B ) human islets, hepatocytes, and HEK293T cells (n = 5–8); ( C ) EndoC-βH1, HepG2, and HEK293T cells (n = 6–7). D and E , detection by Western blot and quantification of intracellular PCSK9 in EndoC-βH1, HepG2, and HEK293T cells (n = 5). F and G , detection by Western blot and quantification of secreted PCSK9 in EndoC-βH1, HepG2, and HEK293T cells (n = 5–6). H , Western blot analysis of PCSK9 secretion by human islets, compared with HEK293T cells (n = 7). Data represent the means ± SD. ∗ p < 0.05 and ∗∗ p < 0.01. FACS, fluorescence-activated cell sorting; HEK293T, human embryonic kidney 293T cell line; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, FACS

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: Cholesterol, LDL and mevastatin modulate PCSK9 expression and secretion. A – F , endoC-βH1 cells were exposed to 10 μg/ml human LDL, 0.25 mM cholesterol–methyl-β-cyclodextrin, and 10 μg/ml mevastatin for 24 h ( A and B ) SREBP-2 transcriptional targets ( HMGCR and LDLR ), SREBP-1 transcriptional targets ( FASN and SCD ), and PCSK9 mRNA were measured by RT–qPCR (n = 5). Intracellular ( C and D ) and secreted ( E and F ) PCSK9 were analyzed and quantified by Western blot (n = 4). G and H , endoC-βH1 cells were exposed to 10 μg/ml native LDL, oxidized LDL, or triglyceride-rich lipoproteins (TRLs) for 24 h. PCSK9 , SREBP-1, and SREBP-2 targets were measured by RT–qPCR (n = 4). Data represent the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. LDL, low-density lipoprotein; PCSK9, proprotein convertase subtilisin/kexin type 9; qPCR, quantitative PCR; SREBP, sterol regulatory element binding protein.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: PCSK9 is regulated by the SREBP-1 and SREBP-2 transcription factors. A – C , endoC-βH1 cells were transfected with control nontarget siRNA (siCtrl), siRNA targeting SREBP-1 + SREBP-2 (siSREBP-1 + siSREBP-2), SREBP-2 (siSREBP-2), or SREBP-1 (siSREBP-1). RT–qPCR for SREBPs, and their targets were performed 3 days later (n = 5). D – G , endoC-βH1 cells were transfected with siRNA, cultured for 72 h, and next treated for 24 h with DMSO (control) or mevastatin before analyses by RT–qPCR (n = 5). Data represent the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. DMSO, dimethyl sulfoxide; PCSK9, proprotein convertase subtilisin/kexin type 9; qPCR, quantitative PCR; SREBP, sterol regulatory element binding protein.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Transfection, Control, Quantitative RT-PCR, Cell Culture, Real-time Polymerase Chain Reaction, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: Insights from omics experiments. EndoC-βH1 cells were transfected with control nontarget siRNA (siControl) or siRNA targeting PCSK9 (siPCSK9). Three days later, cells were profiled by RNA-Seq and proteomics. RNA-Seq data were analyzed with ( A ) ingenuity pathway analysis (IPA) software, for the top enriched canonical pathways, and ( B ) Gene Set Enrichment Analysis (GSEA) software with the Hallmark_Oxidative_Phosphorylation gene set. C , GSIS markers deregulated upon PCSK9 knockdown with FDR <0.05 in RNA-Seq experiment. D , basal (0.5 mM), glucose-stimulated (20 mM), and KCl-stimulated (50 mM) insulin secretions were measured in EndoC-βH1 cells, 3 days following PCSK9 knockdown (n = 6). E , Gene Ontology analysis of cellular components terms enriched among genes and proteins upregulated following PCSK9 knockdown. F – H , Boxplot showing protein expression of top candidate PCSK9 degradation targets, measured on cell protein extracts by DIA and TMT proteomic. I , RT–qPCR validation of selected top differentially expressed genes (n = 10). For GSIS and RT–qPCR analyses, data are presented as means ± SD. For proteomics experiments, median expression and interquartile ranges are shown. + FDR < 0.05, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. DIA, data-independent acquisition; FDR, false discovery rate; GSIS, glucose-stimulated insulin secretion; PCSK9, proprotein convertase subtilisin/kexin type 9; qPCR, quantitative PCR; TMT, tandem mass tag.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Transfection, Control, RNA Sequencing, Software, Phospho-proteomics, Knockdown, Expressing, Quantitative RT-PCR, Biomarker Discovery, Data-independent acquisition, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: PCSK9 knockdown does not affect LDLR levels . A and B , endoC-βH1 cells were exposed for 16 h to human recombinant PCSK9 (2.5 μg/ml). LDLR protein expression was studied by Western blot and quantified (n = 4). C – K , endoC-βH1 cells were transfected with control nontarget siRNA (siCtrl) or siRNA targeting PCSK9 (siPCSK9). Analyses were performed 3 days later. C – F , PCSK9 knockdown efficiency was analyzed by RT–qPCR (n = 9) ( C ) and Western blot for intracellular and secreted PCSK9 (n = 5–7) ( D – F ). G – K , the effect of PCSK9 knockdown on LDLR was measured by RT–qPCR (n = 7), Western blot (n = 5), and FACS for cell surface expression (n = 6). Data represent the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. FACS, fluorescence-activated cell sorting; LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; qPCR, quantitative PCR.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Knockdown, Recombinant, Expressing, Western Blot, Transfection, Control, Quantitative RT-PCR, Fluorescence, FACS, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: PCSK9 knockdown potentiates the induction of PDL1 and HLA-ABC by interferon-γ (IFN-γ). A – D , endoC-βH1 cells were transfected with control nontarget siRNA (siCtrl) or siRNA targeting PCSK9 (siPCSK9). Five days later, the cells were incubated for 24 h with 500 U/ml IFN-γ. A and B , RT–qPCR analysis of PCSK9, PDL1, and MHC-I (n = 6–7). C and D , cell surface PDL1 expression analyzed by FACS (n = 8–9). E and F , endoC-βH1 cells were treated with 500 U/ml IFN-γ. Twenty-four hours later, human recombinant PCSK9 (rPCSK9) (2.5 μg/ml) was added to the culture medium for 16 h. Cell surface PDL1 was studied by FACS (n = 4). G – J , cell surface HLA-ABC expression analyzed by FACS under the same experimental conditions as in ( A – F ) (n = 4–7). Data represent the means ± SD. ∗ p < 0.05 and ∗∗ p < 0.01. FACS, fluorescence-activated cell sorting; MHC-I, major histocompatibility complex; PCSK9, proprotein convertase subtilisin/kexin type 9; qPCR, quantitative PCR.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Knockdown, Transfection, Control, Incubation, Quantitative RT-PCR, Expressing, Recombinant, Fluorescence, FACS, Immunopeptidomics, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits

doi: 10.1016/j.jbc.2022.102096

Figure Lengend Snippet: PCSK9 regulates the expression of VLDLR and CD36, two actors involved in fatty acid homeostasis . A and B, endoC-βH1 cells were transfected with control nontarget siRNA (siCtrl) or siRNA targeting PCSK9. Three days later, VLDLR expression was studied by Western blot and quantified (n = 5–6). C and D, endoC-βH1 cells were exposed for 16 h to human recombinant PCSK9 (2.5 μg/ml), and VLDLR protein expression was studied by Western blot (n = 4). E and F, endoC-βH1 cells were transfected with siRNA (siCtrl) or siRNA targeting PCSK9. Six days later, cell surface CD36 was analyzed by FACS (n = 4). G and H, endoC-βH1 cells were exposed for 16 h to 2.5 μg/ml human recombinant PCSK9, and cell surface CD36 was analyzed by FACS (n = 4). I, fatty acid uptake was evaluated 6 days following PCSK9 knockdown (n =10). Data represent the means ± SD. ∗ p < 0.05 and ∗∗∗ p < 0.001. CD36, cluster of differentiation; FACS, fluorescence-activated cell sorting; PCSK9, proprotein convertase subtilisin/kexin type 9; VLDLR, very low–density lipoprotein receptor.

Article Snippet: The following antibodies were used: PCSK9 (1/200 dilution; catalog no.: AF3888; R&D Systems), diluted in Tris-buffered saline 0.1% Tween with 0.5% bovine serum albumin (BSA); LDLR (1/1000 dilution; catalog no.: ab52818; Abcam), VLDLR (1/1000 dilution; catalog no.: ab75591; Abcam), alpha-tubulin (1/2000 dilution; catalog no.: T9026; Sigma–Aldrich), and beta-actin (1/2000 dilution; catalog no.: A5441; Sigma–Aldrich).

Techniques: Expressing, Transfection, Control, Western Blot, Recombinant, Knockdown, Fluorescence, FACS