Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Generation and characterization of Cre-dependent dCas9-VPR-expressing mice. ( A ) Scheme of the Cre-dependent dCas9-VPR Rosa26 targeting vector. ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre gene (AAV8- Cre ) or 6 different gRNAs against Pcsk9 (AAV8- gPcsk9 ), named gPcsk9-1 to 6 , at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C ) PCR representation showing LSL cassette recombination in liver tissues isolated from Rosa26-LSL-dCas9-VPR mice transduced with AAV8- Cre or AAV8- gPCSK9 alone. A representative agarose gel electrophoresis image is shown. Lane 1–3 contains amplicons obtained from tissue samples of three different mice treated with AAV8- gPcsk9 and lanes 4–6 from tissue samples of three different mice treated with AAV8- Cre . The expected size of PCR products, marker (M) and NTC (H2O) are indicated. ( D ) dCas9-VPR RNA expression in tissues dissected from AAV8- Cre or AAV8- gPCSK9 injected Rosa26-LSL-dCas9-VPR mice. dCas9-VPR expression can only be seen in AAV8- Cre treated Rosa26-LSL-dCas9-VPR mice liver samples. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 10).

Article Snippet: The following primary antibodies were used: LDLR (1:50, PAB8804, Abnova), SERPINA1A (1:20, MAB7690, R&D Systems), PCSK9 (1:10, AF3985, R&D Systems) and β-actin (1:20, NB600-501, Novus Biologicals).

Techniques: Expressing, Plasmid Preparation, In Vivo, Injection, Isolation, Transduction, Agarose Gel Electrophoresis, Marker, RNA Expression, Quantitative Proteomics, Control

Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Parallel Ldlr and Pcsk9 overexpression in vivo using AAV8-transduced CRISPRa mice. ( A ) Relative localization of the 6 individual gRNAs (black bars) targeting mouse Pcsk9 and Ldlr genes are depicted. The transcription start site (TSS) in the promoter regions are shown as black arrows. The numbers indicate the distance in base pair relative to the TSS. The position where individual gRNAs bind to the genome is displayed relative to TSS of the respective Ldlr and Pcsk9 transcripts (Ldlr-201 (ENSMUST00000034713.9), Pcsk9-201 (ENSMUST00000049507.6)). ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre (AAV8- Cre ) alone or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both, at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C , D ) Ldlr and Pcsk9 mRNA expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 9–10). ( E ) Ldlr protein expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 9–10). ( F ) Pcsk9 protein expression in liver tissue isolated from Rosa26-LSL-dCas9-VPR mice were treated with AAV8- Cre alone, or in combination with AAV8- gLdlr , AAV8- gPcsk9 or a mixture of both. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 10). ( G ) PCSK9 protein levels detected in mouse plasma from Rosa26-LSL-dCas9-VPR mice. Means and standard deviations are shown, n = 10. ( H ) Representative images of anti-LDL receptor stained liver sections showing differential LDLR expression in Rosa26-LSL-dCas9-VPR mice injected with AAV- Cre alone, together with AAV- gLdlr or AAV8- gPcsk9 , or simultaneous treatment. Upper panel scale bar, 500 µm. Lower panel, magnified, scale bar, 100 µm.

Article Snippet: The following primary antibodies were used: LDLR (1:50, PAB8804, Abnova), SERPINA1A (1:20, MAB7690, R&D Systems), PCSK9 (1:10, AF3985, R&D Systems) and β-actin (1:20, NB600-501, Novus Biologicals).

Techniques: Over Expression, In Vivo, Injection, Expressing, Isolation, Quantitative Proteomics, Control, Clinical Proteomics, Staining

Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Plasma LDL, HDL and cholesterol concentrations in Rosa26-LSL-dCas9-VPR mice after AAV8- gLdlr/gPcsk9 mediated upregulation of hepatic LDLR and/or PCSK9. ( A , B ) Concentrations of LDL and HDL in plasma from Rosa26-LSL-dCas9-VPR mice. Mean values of concentrations and standard deviations are shown, n = 10. ( C ) Total cholesterol concentrations in plasma from Rosa26-LSL-dCas9-VPR mice. Means and standard deviations are shown, n = 10.

Article Snippet: The following primary antibodies were used: LDLR (1:50, PAB8804, Abnova), SERPINA1A (1:20, MAB7690, R&D Systems), PCSK9 (1:10, AF3985, R&D Systems) and β-actin (1:20, NB600-501, Novus Biologicals).

Techniques: Clinical Proteomics

Journal: Oncology letters

Article Title: Overexpression of circ-0067934 is associated with increased cellular proliferation and the prognosis of non-small cell lung cancer.

doi: 10.3892/ol.2018.9357

Figure Lengend Snippet: Figure 1. Expression of circ‑0067934 is increased in NSCLC tissues and cells. (A) The expression of circ‑0067934 was detected in 79 pairs of tumor tissues and adjacent non‑cancerous tissues using RT‑qPCR. (B) The expression of circ‑0067934 was detected in NSCLC cell lines (A549, H1299, SK‑MES‑1 and PC‑9) and in the normal human bronchial epithelial BEAS‑2B cell line using RT‑qPCR, BEAS‑2B cells represented to control group, the circ‑0067934 expres sion of which was set at a value of 1. *P<0.05 vs. BEAS‑2B or as indicated. circRNA, circular RNA; NSCLC; non‑small cell lung cancer; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

Article Snippet: Human NSCLC cell lines (A549, H1299, SK-MES-1, PC-9) and a normal human bronchial epithelial cell line (BEAS-2B) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Control, Polymerase Chain Reaction

Journal: Oncology letters

Article Title: Overexpression of circ-0067934 is associated with increased cellular proliferation and the prognosis of non-small cell lung cancer.

doi: 10.3892/ol.2018.9357

Figure Lengend Snippet: Figure 2. Expression of circ‑0067934 is associated with the prognosis of patients with NSCLC. The association between circ‑0067934 expression and (A) lymph node metastasis and (B) Tumor‑Node‑Metastasis stage in patients with NSCLC. (C) The association between circ‑0067934 expression and overall survival time in patients with NSCLC was analyzed using Kaplan‑Meier analysis and the log‑rank test. (D) The expression of circ‑0067934 was detected using reverse transcription‑quantitative polymerase chain reaction following treatment with si‑NC, S1 or S2 in A549 and SK‑MES‑1 cells. *P<0.05. circRNA, circular RNA; NSCLC; non‑small cell lung cancer; si/siRNA, small interfering RNA; NC, negative control; S1, si‑circ‑0067934‑1; S2, si‑circ‑0067934‑2; positive, patients with NSCLC with lymph node metastasis; negative, patients with NSCLC without lymph node metastasis.

Article Snippet: Human NSCLC cell lines (A549, H1299, SK-MES-1, PC-9) and a normal human bronchial epithelial cell line (BEAS-2B) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Small Interfering RNA, Negative Control

Journal: Oncology letters

Article Title: Overexpression of circ-0067934 is associated with increased cellular proliferation and the prognosis of non-small cell lung cancer.

doi: 10.3892/ol.2018.9357

Figure Lengend Snippet: Figure 3. Downregulation of circ‑0067934 suppresses the proliferation of NSCLC cells. Cell proliferation was analyzed in (A) A549 and (B) SK‑MES‑1 cells that were treated with si‑NC, S1 or S2 for 24, 48, 72 and 96 h. *P<0.05 vs. si‑NC group. (C) Representative images and (D) numbers of colonies of A549 and SK‑MES‑1 cells that were treated with si‑NC, S1 or S2. *P<0.05. circRNA, circular RNA; NSCLC, non‑small cell lung cancer; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction; si/siRNA, small interfering RNA; NC, negative control; S1, si‑circ‑0067934‑1; S2, si‑circ‑0067934‑2.

Article Snippet: Human NSCLC cell lines (A549, H1299, SK-MES-1, PC-9) and a normal human bronchial epithelial cell line (BEAS-2B) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Polymerase Chain Reaction, Small Interfering RNA, Negative Control

Journal: Developmental cell

Article Title: Programmable Extracellular Vesicles for Macromolecule Delivery and Genome Modifications

doi: 10.1016/j.devcel.2020.11.007

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Constructs pCMV-VSV-G-Myc Neil King/University of Washington N/A pBbsr-VSV-G-sfGFP This paper N/A pBbsr-VSV-G-GFP11 This paper N/A pBbsr-VSV-G-Myc-vpr-GFP11 This paper N/A pBbsr-VSV-G-Myc-vpr-GFP1-10 This paper N/A pCMV-VSV-G-P127D-Myc Neil King/University of Washington N/A pBbsr-VSV-G-P127D-Myc-vpr-GFP11 This paper N/A pBbsr-VSV-G-NJ-GFP11 This paper N/A pBbsr-VSV-G-Myc-Cre This paper N/A pCMV4-BlaM-Vpr Neil King/University of Washington N/A pBbsr-BlaM-Vpr-GFP1-10 This paper N/A pBbsr-Cre-GFP1-10 This paper N/A EGFP-hAgo2 Leung et. al., 2006 Addgene plasmid #21981 pBbsr-AGO2-GFP1-10 This paper N/A pBS-elav-1 Wang et al., 2010 Addgene plasmid #26130 pBbsr-Elav1-GFP1-10 This paper N/A pX602-AAV-TBG::NLS-SaCas9-NLS-HA-OLLOA S-bGHpA;U6::BsaI-sgRNA Ran et al., 2015 Addgene plasmid #61593 pBbsr-NLS-SaCas9-NLS-HA-GFP1-10 This paper N/A pBbsr-CD9-GFP11 This paper N/A pBbsr-CD81-GFP11 This paper N/A Lenti-CRISPR/Cas9-sgMunc13-4 This paper N/A pLKO-PINK1-shRNA Zhang, et al, 2014 N/A pLKO-PCSK9-shRNA This paper N/A LentiCRISPR-EGFP sgRNA 1 Shalem et al., 2014 Addgene plasmid #51760 pEntry-bGH-U6-(SaCas9)-sgPINK1 This paper N/A pEntry-bGH-U6-(SaCas9)-sgEGFP This paper N/A pEntry-bGH-U6-(SaCas9)-sgPCSK9 This paper N/A pEntry-bGH-U6-(SaCas9)-sgRosa26 This paper N/A pBbsr-CD47-Myc-GFP11 This paper N/A pBbsr-CD47nanobody-Myc-GFP11 This paper N/A Cell Lines 293T ATCC Cat#CRL-1573 293ColorSwitch This paper N/A HeLa ATCC Cat#CCL-2 HeLa-Venus-Parkin-RFP-Smac Zhang, et al., 2014 .

Techniques: Construct, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Electron Microscopy, Recombinant, Magnetic Beads, Isolation, In Vivo, Labeling, Flow Cytometry, Software

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a PCSK9 was measured from bronchoalveolar lavage fluid (BALF) of both smokers with ( n = 18) or without chronic obstructive pulmonary disease (COPD) ( n = 17) and non-smokers ( n = 17). b – d Primary bronchial epithelial cells (PBEC) were cultured with PCSK9 or in untreated (UT) condition for 24 h. In response to stimulation with PCSK9, PBEC induced production of pro-inflammatory cytokines, including IL-6, TNF-α, and the chemokine IL-8 ( n = 6). e PCSK9d induced matrix metalloproteinase 9 (MMP9) expression, with a concentration-dependent effect ( n = 6). f PCSK9 was identified as an inducer of phosphorylation of MAPKp38 ( n = 3), with higher concentrations demonstrating a more pronounced effect. Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Cell Culture, Expressing, Concentration Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a ) Uniform Manifold Approximation and Projection (UMAP) representation of human lung single cells grouped into 9 distinct cell types. b Comparison of PCSK9 expression levels amongst healthy and different conditions of lung diseases (Healthy: 4 donors; Pulmonary Fibrosis: 6 donors; Pulmonary Systemic Sclerosis: 5 donors; COPD: 18 donors).

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Comparison, Expressing

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a PCSK9 levels were quantified from cell lysates of A549 cell ( n = 6) and PBEC ( n = 6) by ELISA. b Western blot from the cells lysates of A549 cells and PBEC. c In addition, immunostaining was performed to visualize the microscopic images for the PCSK9, scale bar 10 µm. Both PBEC and A549 cells expressed PCSK9 at the protein level. d Differentiated PBEC on air liquid interface (ALI) exhibited higher levels of PCSK9 protein compared to undifferentiated PBEC in submerged culture (SM). e In compared to untreated (UT) condition, treatment with cigarette smoke extract (CSE) resulted in a reduction of endogenous PCSK9 protein levels in undifferentiated PBEC. f , g Undifferentiated PBEC, upon CSE treatment, released PCSK9 into the cell culture ( n = 6). Notably, this CSE-induced PCSK9 release was inhibited by brefeldin ( n = 6). h In CSE-treated PBEC, PCSK9 was primarily located in the cytoplasm, 10 µm scale bar was used for microscopic images i Minimal increase in PCSK9 was observed in differentiated PBEC in response to CSE. Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Cell Culture

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: Bulk RNA sequencing analysis from control siRNA and PCSK9 siRNA transfected PBEC. a Endogenous inhibition of PCSK9 showed alterations in the expression of approximately 2000 genes, both upregulated and downregulated genes. b , c The impact of PCSK9 inhibition extended to crucial biological pathways and processes, demonstrating an influence on cellular functions. 10 upregulated and 10 downregulated crucial pathways or biological process were presented. d Gene co-expression network of affected genes encompassed pathways and processes vital to cellular function, such as cholesterol synthesis, COX, apoptosis, ARH (Aryl hydrocarbon receptor), and the CYP450 pathway.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: RNA Sequencing, Control, Transfection, Inhibition, Expressing, Cell Function Assay

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a Lipid level was measure by lipid accumulation specific bodipy reagent. Inhibition of PCSK9 was associated with an increase in lipid accumulation. b , c Lipid peroxidation was measured by lipid peroxidation specific bodipy and lipid peroxidation product MDA ( n = 3). In compared to untreated (UT), cigarette smoke extract (CSE) induced a higher level of lipid peroxidation in cells in the PCSK9-suppressed PBEC. d Cell cycle was analyzed by propidium iodide staining. PCSK9 inhibition affected cell cycle, primarily affecting the G2/M phase. e LDH assay was performed to determine cell death. CSE-induced cell death was notably higher in PCSK9-silenced cells compared to control siRNA transfected cells ( n = 6). f , g In response to the staurosporine, PCSK9 inhibition induced apoptosis and an increase in caspase 3 activation ( n = 3). Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Inhibition, Staining, Lactate Dehydrogenase Assay, Control, Transfection, Activation Assay

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: A In response to xenobiotics PCSK9 transport from nucleus to Golgi through endoplasmic reticulum and transport vacuoles (1-2). Damaged cell membranes allow the PCSK9 release outside the cells (3) and stimulate other cells to induce MAPK activation and release cytokines (4a). Additionally, PCSK9 alter lipid hemostasis (4b). B In absence of PCSK9, cell cycle alteration results reduced proliferation (1) and in that condition exposure to xenobiotic induces cell death (2) Additionally, in absence of PCSK9 lipid peroxidation (1b) may induce ferroptosis (2b). The image was generated by using smart servier medical art tools. ( https://smart.servier.com/citation-sharing ). The elements were remixed and incorporated with power point elements.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Activation Assay, Generated

Journal: Journal of Lipid Research

Article Title: PCSK9 is not secreted from mature differentiated intestinal cells

doi: 10.1016/j.jlr.2021.100096

Figure Lengend Snippet: Caco2 cells display a biphasic secretion of PCSK9 at the basolateral side during their differentiation into enterocytes. Caco2 cells were maintained on polycarbonate micropore membranes inserted into Transwells. The medium was changed every two days. A and B: Basolateral supernatants were collected every two days from day 4 to day 22 in two or more independent experiments done in triplicates. A: PCSK9 was measured in supernatants by ELISA. B: Proteins from supernatants (300 μl) were precipitated with acetone, and apoB and PCSK9 were analyzed by Western Blot (WB). C: Caco2 cells were recovered at day 8 (d8) and at day 16 (d16) after a 2-h secretion assay, and mRNAs were extracted. Sucrase isomaltase and ApoB expression relative to that of cyclophilin was analyzed by RT-qPCR in samples from nine independent experiments. Scatter dot plots + bars (median ± interquartile) are presented. ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. D and E: Day 8 (d8) and day 16 (d16) after Caco2 cells seeding, the PCSK9 content in supernatants from basolateral and apical sides after a secretion assay of 2 h was measured (C) by ELISA, scatter dot plots + histograms represent median ± interquartile. ∗∗ P value < 0.01, or analyzed (D) by WB after acetone protein precipitation, the right blot showing apical PCSK9 secretion has been voluntarily overexposed to facilitate band visualization. Results are from six and three independent experiments, respectively. PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: We used monoclonal or polyclonal antibodies raised against either human PCSK9 (ab181142 from Abcam, Paris, France/AF3888 from R&D Systems), mouse/rat PCSK9 (AF3995 from R&D Systems), human apoB (AB742 from Merck Millipore), human or mouse beta actin (A5616 or A5451, respectively, from Sigma-Aldrich), and suitable HRP-secondary antibodies purchased from Cell Signaling Technology or R&D systems.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: PCSK9 is not secreted from mature differentiated intestinal cells

doi: 10.1016/j.jlr.2021.100096

Figure Lengend Snippet: Intracellular PCSK9 content in Caco-2 cells decreases with cell maturation. Differentiated Caco2 cells at d8 and at d16 were recovered after a 2-h secretion assay, and intracellular proteins were extracted from cells. A: Intracellular PCSK9 content (ng extracted from cells) in samples from six independent experiments were measured by ELISA. B: Intracellular pro-PCSK9, PCSK9 and β-actin contents were analyzed by WB. Representative images from three independent experiments are shown (left panel). Pro-PCSK9 and PCSK9 contents relative to b-actin content from seven independent experiments are shown (right panel). C: Cells lysates were denatured and incubated 3 h at 37°C in presence or not of endoH or PNGase F enzymes, followed by WB for PCSK9. Bands corresponding to undigested Pro-PCSK9 (Pro-PCSK9), undigested PCSK9 (PCSK9), digested PCSK9 (Dig-PCSK9), and digested Pro-PCSK9 (Dig-Pro-PCSK9) by enzymes are indicated. One representative result from two independent experiments is shown. Histograms represents the median ± interquartile. ∗ P value< 0.05, ∗∗∗ P value <0.001. PCSK9, proprotein convertase subtilisin/kexin type 9; WB, Western blot.

Article Snippet: We used monoclonal or polyclonal antibodies raised against either human PCSK9 (ab181142 from Abcam, Paris, France/AF3888 from R&D Systems), mouse/rat PCSK9 (AF3995 from R&D Systems), human apoB (AB742 from Merck Millipore), human or mouse beta actin (A5616 or A5451, respectively, from Sigma-Aldrich), and suitable HRP-secondary antibodies purchased from Cell Signaling Technology or R&D systems.

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

Journal: Journal of Lipid Research

Article Title: PCSK9 is not secreted from mature differentiated intestinal cells

doi: 10.1016/j.jlr.2021.100096

Figure Lengend Snippet: SREBP2-regulated genes, including its own gene SREBF2 and PCSK9 , are downregulated in mature Caco-2 cells. Caco2 cells were recovered at d8 and at d16 after a 2-h secretion assay, and mRNAs were extracted. A: PCSK9 expression relative to that of cyclophilin was analyzed by RT-qPCR in samples from 9 independent experiments. Scatter dot plots + bars (median ± interquartile) are presented. ∗∗ P < 0.01. B: 3′ Digital gene expression RNA sequencing was performed in samples from 4 independent experiments (E1 to E4) at d8 and d16. MA plot is shown in the left panel: dots corresponding to genes that are significantly upregulated at day 16 (GENES UP), downregulated at day 16 (GENES DOWN), or unchanged are colored in orange, blue, and gray, respectively. A heat map with differentially expressed genes (DEGs) is shown in the right panel: each column represents one sample showing the intensity of the expression profile per gene. C: Bar chart of functional GOBP (gene ontology biological process) terms enrichment analysis for GENES UP and for GENES DOWN is shown. D: Protein-protein interactions for the downregulated genes belonging to the cluster “Cholesterol biosynthetic process” are shown. Line thickness indicates the strength of data support. Four clusters with connected nodes are distinguished according to color of bubbles. E: Log2(fold change) for each downregulated gene of the cluster “Cholesterol biosynthetic process” is indicated. F, SREBF2, HMGCR, and LDLR expression relative to that of cyclophilin was analyzed by RT-qPCR in samples from nine independent experiments. Scatter dot plots + bars (median ± interquartile) are presented. ∗ P < 0.05. HMGCR, HMG-COA reductase; LDLR, LDL receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: We used monoclonal or polyclonal antibodies raised against either human PCSK9 (ab181142 from Abcam, Paris, France/AF3888 from R&D Systems), mouse/rat PCSK9 (AF3995 from R&D Systems), human apoB (AB742 from Merck Millipore), human or mouse beta actin (A5616 or A5451, respectively, from Sigma-Aldrich), and suitable HRP-secondary antibodies purchased from Cell Signaling Technology or R&D systems.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, RNA Sequencing, Functional Assay, Protein-Protein interactions

Journal: Journal of Lipid Research

Article Title: PCSK9 is not secreted from mature differentiated intestinal cells

doi: 10.1016/j.jlr.2021.100096

Figure Lengend Snippet: Human intestinal explants do secrete ApoB but not PCSK9. The epithelial layer from human colon biopsies were mounted in Ussing chambers and incubated for 2 h in an oxygenated Krebs solution at 37°C. After incubation, basolateral and apical media and tissues were collected. A: PCSK9 and (B) ApoB protein levels were measured by ELISA. Scatter dot plots + bars represent the median ± interquartile (two biopsies per patient, five patients in total). PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: We used monoclonal or polyclonal antibodies raised against either human PCSK9 (ab181142 from Abcam, Paris, France/AF3888 from R&D Systems), mouse/rat PCSK9 (AF3995 from R&D Systems), human apoB (AB742 from Merck Millipore), human or mouse beta actin (A5616 or A5451, respectively, from Sigma-Aldrich), and suitable HRP-secondary antibodies purchased from Cell Signaling Technology or R&D systems.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Lipid Research

Article Title: PCSK9 is not secreted from mature differentiated intestinal cells

doi: 10.1016/j.jlr.2021.100096

Figure Lengend Snippet: Comparison of circulating PCSK9 concentrations in several biological compartments. A: PCSK9 concentrations in plasma collected from the tail or portal or mesenteric lymph, bile, urine, and intestinal lumen from C57BL6/J female mice were measured by ELISA. B: PCSK9 concentrations in plasma from cheek vein blood and portal vein blood from PCSK9 LivKO mice or control floxed male and female mice. Scatter dot plots + bars represent the median ± interquartile. ∗∗∗ P < 0.001 versus cheek vein blood in males. §§ P < 0.01 versus portal vein blood in males. PCSK9 LivKO, mouse model lacking PCSK9 specifically in hepatocytes; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: We used monoclonal or polyclonal antibodies raised against either human PCSK9 (ab181142 from Abcam, Paris, France/AF3888 from R&D Systems), mouse/rat PCSK9 (AF3995 from R&D Systems), human apoB (AB742 from Merck Millipore), human or mouse beta actin (A5616 or A5451, respectively, from Sigma-Aldrich), and suitable HRP-secondary antibodies purchased from Cell Signaling Technology or R&D systems.

Techniques: Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control

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: 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.

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: 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.

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: 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.

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: 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.

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

Journal: Cell reports

Article Title: PUF60 promotes cell cycle and lung cancer progression by regulating alternative splicing of CDC25C

doi: 10.1016/j.celrep.2023.113041

Figure Lengend Snippet: (A–C) Effects of PUF60 silencing on cell proliferation (A), cell cycle progression (B), and apoptosis (C) of LUAD A549 and PC9 cells. PUF60 was knocked down using two different small interfering RNAs (siRNAs), and the knockdown efficiency was assessed by western blot analysis, as shown in the top panel in (A). The second siRNA with higher knockdown efficiency was used in (B) and (C). n = 3–6 biological replicates. (D and E) Effects of CRISPR-Cas9-mediated PUF60 depletion (sgPUF60) on cell proliferation (D) and colony formation (E) of LUAD A549 cells. Three sublines were derived from distinct cell colonies (C1, C2, and C3, respectively), where the efficiency of PUF60 depletion was measured by western blot, as shown in the top panel in (D). n = 3 biological replicates. Scale bar: 1 cm. (F and G) Growth curve (F) and the endpoint illustration (G) of xenograft tumors following subcutaneous injection of A549 cells with or without PUF60 depletion in nude mice. Error bar: ± SD; n = 10 in each group; scale bar: 1 cm. Error bars represent ±/+ SEM unless indicated otherwise. *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA with Tukey’s test in (A), (D), and (F), Student’s t test in (B) and (C), and one-way ANOVA with Dunnett’s multiple comparison test in (E).

Article Snippet: PC9 RNA-Seq , This Paper and The National Omics Data Encyclopedia (NODE) database , Accession number: OEP004324.

Techniques: Knockdown, Western Blot, CRISPR, Derivative Assay, Injection, Comparison

Journal: Cell reports

Article Title: PUF60 promotes cell cycle and lung cancer progression by regulating alternative splicing of CDC25C

doi: 10.1016/j.celrep.2023.113041

Figure Lengend Snippet: (A) Volcano plot of alternative splicing (AS) changes following PUF60 knockdown in LUAD A549 and PC9 cells (false discovery rate [FDR] < 0.05, |ΔPSI| > 0.1). The cell cycle genes involved in S and G2/M phases were highlighted. PSI, percent-spliced-in. (B) The numbers of altered splicing events in each AS category. SE, skipped exon; A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; MEX, mutually exclusive exon; RI, retained intron. (C) Gene Ontology (GO) analysis of genes with altered splicing following PUF60 knockdown. BP, biological process. Cell cycle-related GO terms were marked in red. (D) Heatmap illustration of AS changes in cell cycle genes following PUF60 knockdown in LUAD A549 and PC9 cells. AS events were ranked by ascending PSI changes in A549 cells. All AS events were skipped exons. CrE, crypt exon. (E) RT-PCR validation of AS events induced by PUF60 knockdown in LUAD cells. Shown are representative agarose gel images (bottom panel) and quantification of exon inclusion levels (top panel) for four genes. Error bars represent ± SEM; n = 2–3 biological replicates; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: PC9 RNA-Seq , This Paper and The National Omics Data Encyclopedia (NODE) database , Accession number: OEP004324.

Techniques: Alternative Splicing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Agarose Gel Electrophoresis, Comparison

Journal: Cell reports

Article Title: PUF60 promotes cell cycle and lung cancer progression by regulating alternative splicing of CDC25C

doi: 10.1016/j.celrep.2023.113041

Figure Lengend Snippet: (A) Schematic illustration of the splice variants produced from alternative splicing of CDC25C exon 3. Five known CDC25C splice variants (V1–V5) and the splice variant lacking exon 3 only that is predicted to be an NMD substrate were shown. Arrows indicate RT-PCR primers used for detecting those splice variants. PTC, premature termination codon; NMD, nonsense-mediated mRNA decay. (B) RT-PCR and western blot analysis of CDC25C splice variants in LUAD A549 and PC9 cells. PSI, percent-spliced-in; IB, immunoblotting. Loading controls: GAPDH for RT-PCR and β-actin for IB. (C) RT-PCR analysis of CDC25C splice variants following UPF1 and PUF60 knockdown alone or in combination in LUAD A549 cells. NC, non-targeting control. (D) Effects of PUF60 silencing on the splicing of CDC25 exon 3 examined by splicing minigene reporter assays. Top panel: scheme of minigene reporter for CDC25C exon 3 and RT-PCR primers. PUF60 knockdown efficiency was examined by RT-PCR using a primer within its coding sequence region. Loading control: GAPDH. (E) Effects of PUF60 wild type (WT) and mutants (MUTs) on CDC25C exon 3 inclusion. Top panel: schematic illustration of PUF60 domain structure. RRM, RNA recognition motif; UHM, U2AF-homology motif. Bottom panel: splicing outcomes of CDC25C exon 3 from minigene splicing reporter under PUF60 silencing and rescuing with PUF60 WT or MUTs, respectively. Representative agarose gel image and quantification results are shown. Loading control: GAPDH. PSI, percent-spliced-in. Error bars represent ± SEM; n = 2 biological replicates; ns, not significant, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test. (F) Crosslinking and RNA immunoprecipitation (CLIP) followed by RT-PCR confirmation of the interactions between PUF60 and CDC25C pre-mRNA in A549 cells where FLAG-tagged PUF60 was inducibly expressed by doxycycline treatment. Left panel: western blot analysis of IP efficiency. Right panel: scheme of RT-PCR primers and the detection of PUF60 pre-mRNA obtained from CLIP. (G and H) Cell proliferation of PUF60 -silenced and control LUAD A549 cells with or without CDC25C-FL overexpression (G) and CDC25C-Δ E 3 knockdown (H), respectively. Expression of PUF60 and CDC25C under conditions in (G) and (H) were examined by western blot. Loading control: β-actin. Error bars represent ± SEM; n = 3 biological replicates in (G) and n = 2 in (H); *p < 0.05, ***p < 0.001; two-way ANOVA with Tukey’s test.

Article Snippet: PC9 RNA-Seq , This Paper and The National Omics Data Encyclopedia (NODE) database , Accession number: OEP004324.

Techniques: Produced, Alternative Splicing, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Control, Sequencing, Agarose Gel Electrophoresis, Comparison, RNA Immunoprecipitation, Over Expression, Expressing

Journal: Cell reports

Article Title: PUF60 promotes cell cycle and lung cancer progression by regulating alternative splicing of CDC25C

doi: 10.1016/j.celrep.2023.113041

Figure Lengend Snippet: (A) RT-PCR and western blot analysis of CDC25C expression in LUAD A549 and PC9 cells transfected with non-target control siRNA (siNC) or siRNAs selectively targeting splice variants of CDC25C that contain or lack exon 3 (si-E3 and si-Δ E 3) or targeting the entire gene (si-All), respectively. Loading controls: GAPDH for RT-PCR and β-actin for IB. IB, immunoblotting. Schemes of the siRNAs and RT-PCR primers are shown in the top panel. (B) Cell proliferation under conditions described in (A). (C) RT-PCR and western blot analysis of CDC25C expression in LUAD A549 and PC9 cells transfected with non-target control antisense oligonucleotide (ASO-Ctrl), or ASOs targeting the 3′ splice sites of CDC25C exon 3 (ASO-3ss). Schemes of the ASOs were shown in the upper panel. (D) Cell proliferation following ASO-mediated blockade of CDC25C exon 3 inclusion in LUAD A549 and PC9 cells, respectively. Loading controls: GAPDH for RT-PCR, and β-actin for IB. (E) Scheme of mRNA and protein domain of CDC25C-FL and the short splice variant lacking exons 3, 5, and 6 (CDC25C-S), respectively. (F) Cell proliferation following doxycycline-induced overexpression of CDC25-FL or CDC25-S in LUAD A549 cells, respectively. Inset: CDC25C protein expression examined by western blot. Loading control: β-actin. Dox, doxycycline. Error bars represent ± SEM; n = 2 biological replicates in (B) and (D) and n = 3 in (F); *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant; two-way ANOVA with Tukey’s test in (B), (D), and (F).

Article Snippet: PC9 RNA-Seq , This Paper and The National Omics Data Encyclopedia (NODE) database , Accession number: OEP004324.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Control, Variant Assay, Over Expression

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a , Schematic outline of the architecture of PCSK9 EEs b , Primary screen evaluating 240 candidate gRNAs targeting the human PCSK9 gene using a spCas9-based EE. Each point represents the average of two independent measurements of secreted PCSK9 protein levels 7 d after transfection; the location of each point along the x axis indicates the position of the gRNA relative to the distance (in nucleotides) to the PCSK9 gene TSS. PCSK9 protein levels in cells transfected with a non-targeting (NT) gRNA or effector only (no gRNA) are shown with a dotted line. CpG location (first row); methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus measured by whole-genome bisulfite sequencing (WGBS) or hybrid capture in neurons (second row); HeLa cells (third row); liver hepatocytes (fourth row); and DNaseI accessibility in human liver (fifth row) are shown below the graph and mapped onto the CpG island (CGI) and PCSK9 5′ gene region. c , The top 40 gRNA were selected from b and were evaluated for their ability to potently and durably silence PCSK9 in HeLa cells for up to 28 d. Individual data points and means are shown ( n = 2 replicates per experimental condition). Results are expressed as percent of secreted PCSK9 protein in cells treated with transfection (txn) reagent only. d , The top five gRNAs were selected based on their activity and durability in HeLa cells (from c ) as well as having full cross-reactivity with the cynomolgus macaque PCSK9 gene. PHHs isolated from PXB mice were treated with LNPs containing selected gRNAs and EE mRNA. Results are shown as mean ± s.d. ( n = 4 replicates per gRNA). For b and c , WT Cas9 served as a control for durable silencing of PCSK9 ; for c and d , CRISPRi served as a control for non-durable silencing of PCSK9 .

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Transfection, Methylation, Methylation Sequencing, Activity Assay, Isolation, Control

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: Top 5 gRNA selected based on their activity and durability in HeLa cells (from Fig. ), as well as having full cross-reactivity with the cynomolgus macaque PCSK9 gene were evaluated for their ability to maintain PCSK9 protein reduction in the presence of 1 mM simvastatin or DMSO (Vehicle). Simvastatin or DMSO was added in the last 24 hrs of incubation. Individual data points and means are shown, n = 2 replicates per experimental condition. Results are expressed as % of secreted PCSK9 protein in cells treated with transfection (txn) reagent only. WT Cas9 serves as a control for durable silencing of PCSK9 .

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Activity Assay, Incubation, Transfection, Control

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a . CpG methylation levels in livers from untreated transgenic mice carrying the human PCSK9 genomic locus ( PCSK9 -Tg, in blue) and primary human hepatocytes (in red). b . Location of top 5 gRNAs is shown relative to the distance (in nucleotides) to the PCSK9 gene transcription start site (TSS). c . Effect of a single administration at near-saturating dose (0.75 mg/kg) of an LNP formulation evaluating an epigenetic editor with individual or combination of two gRNA candidates on circulating PCSK9 protein levels in PCSK9 -Tg. Results are shown as mean ± s.d. (n = 6 mice/group). d . Effect of a single administration at a sub-saturating dose (0.2 mg/kg) of an LNP formulation evaluating epigenetic editor with best individual or combination of two gRNA candidates on circulating PCSK9 protein levels in PCSK9 -Tg mice. Results are shown as mean ± s.d. (n = 4 mice in gRNA41 group and n = 5 mice in gRNA41 + 49 group).

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: CpG Methylation Assay, Transgenic Assay, Formulation

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a . Activity of the LNP formulation with the top-ranked PCSK9 EE (PCSK9-EE) in PHHs isolated from chimeric mice with a humanized liver. Each point represents the average of three independent measurements of secreted PCSK9 protein levels at baseline and at 6 d and 15 d after treatment. Results are shown as mean ± s.d. PHHs treated with APOE only or LNP formulation containing the effector without gRNA (effector only) served as negative controls. b – f , Assessment of the specificity of PCSK9-EE was performed using PHHs obtained at 15 d after treatment. b , Specificity testing was assessed using RNA-seq on three independent replicates of each control condition (ApoE Only and Effector Only) and PCSK9-EE. On-target PCSK9 TPM from RNA-seq for each replicate are shown in the dot plot. c , Volcano plot of RNA-seq data comparing PCSK9-EE versus Effector Only control. Thresholds for differential expression: adjusted P value (DEseq2 Wald test, two-sided, Benjamini–Hochberg multiple comparisons adjustment) < 1×10 −5 , log 2 FC > 1 or log 2 FC < −1. PCSK9 is shown as a yellow circle; off-target (upregulated) DEG is shown as a navy blue circle; all other genes below the thresholds are shown as light blue circles. d , Specificity of methylation at CpG-enriched sites was measured using a Twist Human Methylome Hybrid Capture Methylation Sequencing assay. Volcano plot of CpG methylation comparing PCSK9-EE versus Effector Only control. Individual CpGs are colored according to whether they were called as a DMR at the PCSK9 locus (yellow), at an off-target genomic region (navy blue) or were not part of a DMR (light blue). DMR thresholds were set as P value (DSS Wald test, two-sided, unadjusted) < 1 × 10 −10 , beta value difference < −0.2 or beta value difference > 0.2. e , Manhattan plot of genome-wide methylation, as determined by a WGMS assay, comparing PCSK9-EE versus Effector Only control. Benjamini–Hochberg (false discovery rate (FDR)) adjusted P values for each CpG (DSS Wald test, two-sided) are plotted versus genomic coordinate for each CpG. Differentially methylated CpGs within the PCSK9 DMR are shown in yellow. The DMR threshold was set as P value (DSS Wald test, two-sided, unadjusted) < 1 × 10 −10 . f , Scatterplot showing methylation difference of DMRs from WGMS ( y axis) versus log 2 FC from RNA-seq ( x axis) of all genes within 20 kb of each DMR for the PCSK9-EE versus Effector Only control comparison. PCSK9 gene is shown in yellow. Thresholds (gray dashed lines) are set as methylation (beta value) difference > 0.2 or < −0.2, RNA-seq log 2 FC > 1 or < −1. DEG, differentially expressed gene.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Activity Assay, Formulation, Isolation, RNA Sequencing, Control, Quantitative Proteomics, Methylation, Sequencing, CpG Methylation Assay, Genome Wide, Comparison

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a . WGMS CpG methylation for Effector Only (grey) and PCSK9-EE (blue) in the genomic region surrounding off-target DEG ENSG00000285976 (boxed gene) (left) and low level of baseline expression (TPM) of off-target DEG ENSG00000285976 from RNA-Seq is shown in the dot plot (n = 3) (right). b . Scatterplot of RNA-Seq Log2 fold-change of all genes within 20 kb of any DMR called in Twist Human Methylome Hybrid Capture Methylation Sequencing assay (PCSK9-EE vs Effector Only) vs methylation difference of the associated DMR for each gene (PCSK9-EE vs Effector Only). PCSK9 gene/DMR is shown in yellow. Thresholds (grey dashed lines) set as methylation (beta-value) difference > 0.2 or < -0.2, RNA-Seq Log2FC > 1 or < -1. c-i . For all DMRs meeting either the methylation or gene expression change thresholds in Fig. and Extended Data Fig. 3b, WGMS CpG methylation for Effector Only (grey) and PCSK9-EE (blue) in the genomic region surrounding each off-target DMR (left) and expression (TPM) of all off-target genes with transcription start sites within 20 kb of each DMR from RNA-Seq is shown in the dot plot (n = 3) (right). RNA-seq padj values are from a DEseq2 Wald test, two-sided, with Benjamani-Hotchburg multiple comparison correction. c . DMR near WDR81/MIR22HG/SERPINF2 , average methylation difference within DMR by Twist Human Methylome assay: 0.24. Average methylation difference within DMR by WGMS: 0.18. WDR81 RNA-Seq padj = 9.998728e-01, MIR22HG RNA-Seq padj = 9.998728e-01, SERPINF2 RNA-Seq padj = 5.354105e-02, d . DMR near RB1-DT/PPP1R26P1 , average methylation difference within DMR by WGMS: 0.14. RB1-DT RNA-Seq padj = 9.998728e-01, PPP1R26P1 RNA-Seq padj = 9.998728e-01. e . DMR near MCF2 , average methylation difference within DMR by Twist Human Methylome assay: 0.07, MCF2 RNA-Seq padj = 9.998728e-01, f . DMR near TCEA1P3 , average methylation difference within DMR by Twist Human Methylome assay: -0.16, TCEA1P3 RNA-Seq padj = 9.998728e-01, g . DMR near SLC47A2 , average methylation difference within DMR by WGMS: 0.13, SLC47A2 RNA-Seq padj = 9.998728e-01. h . DMR near L3MBTL1 , average methylation difference within DMR by Twist Human Methylome assay: −0.22, L3MBTL1 RNA-Seq padj = 9.998728e-01, i . DMR near Y_RNA, average methylation difference within DMR by WGMS: 0.11, Y_RNA RNA-Seq padj = 9.998728e-01.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: CpG Methylation Assay, Expressing, RNA Sequencing, Methylation, Sequencing, Gene Expression, Comparison

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a , Schematic outline of the in vivo study in transgenic mice ( PCSK9 -Tg) carrying the human PCSK9 genomic locus. The mice were treated with LNPs formulated with the top-ranked PCSK9 EE (PCSK9-EE), CRISPRi or WT Cas9 payload. Illustration was created with BioRender. b , Circulating PCSK9 protein levels in PCSK9 -Tg over a 1-year period after a single administration of an LNP formulation with PCSK9-EE ( n = 6 mice per group). c – e , Effect of a single administration of an LNP formulation with PCSK9-EE on PCSK9 mRNA ( c ), total plasma cholesterol ( d ) and CpG methylation levels in liver ( e ) in PCSK9 -Tg mice 1 month after treatment ( n = 4 mice per group). For e , liver methylation data from all PCSK9 -Tg mice treated with PCSK9-EE for 1 year ( n = 6, from b ) are also included. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus as measured by WGBS in cells expressing PCSK9 (liver hepatocyte WGBS; second row) or not expressing PCSK9 (neuron WGBS; third row) are shown. Vehicle-treated animals received a single administration of saline solution. CRISPRi served a control for robust but transient silencing of PCSK9 , whereas WT Cas9 served as a control for durable silencing of PCSK9 . For b – d , results are shown as mean ± s.d. For c and d , statistical analysis was performed by one-way ANOVA followed by two-tailed Dunnett’s test. For c , *** P = 0.000893; **** P = 000005 versus vehicle-treated mice. For d , *** P = 0.000253; **** P = 0.000024 versus vehicle-treated mice. For e , CpG methylation profiles for all analyzed samples are shown.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: In Vivo, Transgenic Assay, Formulation, Clinical Proteomics, CpG Methylation Assay, Methylation, Expressing, Saline, Control, Two Tailed Test

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a , Schematic outline of the in vivo PHx study. The timing of the PHx (or sham) procedure and the length of time allowed for full liver regeneration before liver sample collection are highlighted. Illustration was created with BioRender. b , Circulating PCSK9 protein levels after a single administration of an LNP formulation with the top-ranked PCSK9 EE (PCSK9-EE) in PCSK9 -Tg mice before and after PHx ( n = 6) or sham ( n = 5) procedures. The PHx or sham procedure was performed on day 35. WT Cas9 served as a control for durable silencing before and after PHx ( n = 6) or sham ( n = 6) procedures. Control animals received saline (vehicle) and were also subjected to pre-PHx and post-PHx ( n = 8) or sham ( n = 3) procedures. c , Effect of a single administration of an LNP formulation with PCSK9-EE on CpG methylation levels in liver from PCSK9 -Tg mice at 90 d after LNP treatment. Methylation data from the resected liver section after the PHx procedure at day 35 were also included in the analysis. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus in cells expressing PCSK9 (liver hepatocyte; second row) or not expressing PCSK9 (neuron; third row) are shown. For b , results are shown as mean ± s.d. For c , CpG methylation profiles for all analyzed samples are shown.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: In Vivo, Formulation, Control, Saline, CpG Methylation Assay, Methylation, Expressing

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a , Schematic outline of PCSK9 silencing in mice using a PCSK9 EE (PCSK9-EE), followed at more than 100 d by treatment with a PCSK9 activator (dCas-Tet). Illustration was created with BioRender. b , Circulating PCSK9 protein levels after a single administration of an LNP formulation with dCas-Tet in PCSK9 -Tg previously treated with PCSK9-EE to silence PCSK9 . Results are shown as mean ± s.d. ( n = 5 mice). c , Effect of a single administration of LNP formulation with dCas-Tet in PCSK9 -Tg mice previously treated with PCSK9-EE to silence PCSK9 on CpG methylation levels at 56 d after dCas-Tet treatment. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus in cells expressing PCSK9 (liver hepatocyte; second row) or not expressing PCSK9 (neuron; third row). CpG methylation profiles for all analyzed samples are shown for c .

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Formulation, CpG Methylation Assay, Methylation, Expressing

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: a , In silico alignment of CpGs (the molecular target of the EE) between human and cynomolgus monkey around the PCSK9 TSS. Matched and unmatched CpGs are labeled in blue and yellow, respectively (84 out of 112 CpGs are matched). Note that the cynomolgus PCSK9 gene is located on the negative strand; hence, CpGs in the reverse complement (rev-comp) sequence are shown. b , Activity and potency of LNP formulation using the EE PCSK9-EE-V2 in cultured PHHs and PCHs were assessed by measuring secreted PCSK9 protein in the supernatant. IC 50 values are indicated. Results are shown as mean ± s.d. ( n = 4 replicates per group). c , Dose–response of a single infusion of an LNP formulation with PCSK9-EE-V2 on circulating PCSK9 protein levels (left) and LDL-C (right) in cynomolgus macaques ( n = 3 per group). Vehicle-treated animals received a single infusion of saline solution ( n = 4). Results are shown as mean ± s.d. Plasma samples were obtained from two of the vehicle-treated animals at days 84 and 98. These data were averaged and plotted at day 91 to better visualize the group mean. d , Effect of a single administration of PCSK9-EE-V2 on CpG methylation levels in liver biopsy samples at 24 d after treatment. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the cynomolgus PCSK9 locus for individual animals are shown. Note that liver biopsies were obtained for only two of the vehicle-treated animals shown in c . e , Pearson’s correlation ( r ) comparing average cynomolgus PCSK9 (c PCSK9 ) TSS methylation levels (day 24) versus cynomolgus PCSK9 protein levels (day 21) for individual animals shown in c and d . Two-sided P value is shown. Cyno, cynomolgus; IC 50 , half-maximal inhibitory concentration.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: In Silico, Labeling, Sequencing, Activity Assay, Formulation, Cell Culture, Saline, Clinical Proteomics, CpG Methylation Assay, Methylation, Concentration Assay

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: Serial measurements of blood alanine transaminase ( a ), aspartate transaminase ( b ), and total bilirubin ( c ) were performed in non-human primates following dosing with either a vehicle control or PCSK9-EE-V2. Results are shown as mean ± s.d. (n = 4 animals in vehicle group and n = 3 animals in each experimental group receiving PCSK9-EE-V2). Plasma samples were obtained from two of the vehicle-treated animals at day 84 and 98. These data have been averaged and plotted at day 91 to better visualize the group mean.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Control, Clinical Proteomics

Journal: Nature Medicine

Article Title: A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: Time-course and dose-response effect of a single infusion of an LNP formulation with PCSK9-EE-V2 on circulating PCSK9 protein levels ( b-d ) and LDL-cholesterol ( g-i ) in individual cynomolgus macaque. Vehicle-treated animals receive a single infusion of saline solution ( a and f ). Average cPCSK9 ( e ) and LDL-cholesterol ( j ) change at day 91 post dose from baseline. For each experimental group, individual animals are represented by a different symbol ( a-j ). For e and j , results are shown as mean ± s.d. (n = 4 animals in vehicle group and n = 3 animals in each experimental group receiving PCSK9-EE-V2).

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Formulation, Saline