Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: Targeted and robust hepatic gene transduction of GFP utilizing AAV8.P-GFP and AAV8.P-PCSK9 shRNA vectors (A) Representative images of GFP expression in various tissues obtained through laser confocal microscopy, assessed 4 weeks post systemic administration of AAV-GFP and AAV-PCSK9 shRNA vectors, scale bar indicates 100 μm. (B) Quantitative analysis of GFP mRNA expression performed via real-time PCR across different tissues at 4 weeks following injection of either AAV-GFP or AAV-PCSK9 shRNA vectors. n = 5/group. Data are shown as means ± SEM. Unpaired t test. (C) Detection of GFP expression through western blot analysis in assorted tissues 4 weeks subsequent to gene transfer. Abbreviations: Li, Liver; He, Heart; Sp, Spleen; Lu, Lung; Ki, Kidney; Br, Brain; Mu, Muscle. (D and E) Western blot assessment of GFP expression specifically in the liver 4 weeks following gene transfer. n = 4/group. Data are shown as means ± SEM. Unpaired t test.

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Transduction, shRNA, Expressing, Confocal Microscopy, Real-time Polymerase Chain Reaction, Injection, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: Silencing hepatic PCSK9 leads to decreased serum levels of total cholesterol and triglycerides, without altering AST or ALT concentrations in C57BL/6J mice (A) Hepatic PCSK9 mRNA levels were quantified using real-time PCR 1-month post-administration of either AAV-GFP or AAV-PCSK9 shRNA, n = 4 for the AAV-GFP cohort and n = 7 for the AAV-PCSK9 shRNA cohort. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (Unpaired t test). (B) Plasma PCSK9 concentrations were assessed through ELISA at 1 and 3 months following transduction with either AAV-GFP or AAV-PCSK9 shRNA, n = 5/group. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (C and D) Serum levels of total cholesterol and triglycerides were determined 1 month and 3 months subsequent to vector administration, n = 8/group. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (E) Serum ApoB concentrations (μg/mL) in C57BL/6J mice were measured 1 month after vector administration, n = 6/group. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (Unpaired t test). (F and G) Immunoblot analysis was conducted for hepatic PCSK9, LDL-R, ApoE, and ApoB-100 1 month after vector administration, with corresponding quantification provided in the bar graph, n = 5/group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01 (Unpaired t test). (H and I) Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were evaluated at 1 and 3 months post-transduction with either AAV-GFP or AAV-PCSK9 shRNA, n = 8/group. Data are shown as means ± SEM (two-way ANOVA with Bonferroni correction).

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, shRNA, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: PCSK9 silencing in ApoE −/− mice induces early changes in lipid profiles but lacks a sustained impact on circulating lipids (A) Hepatic mRNA levels of PCSK9 were quantified using real-time PCR in liver specimens collected 2 weeks post-administration, n = 4/group. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (Unpaired t test). (B) Plasma levels of PCSK9 protein were evaluated via ELISA at baseline and at 2 weeks, 4 weeks, and 6 months following injection, n = 10 for AAV-GFP, n = 12 for AAV-PCSK9 shRNA. Data are shown as means ± SEM. ∗∗ p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (C and D) Serum concentrations of total cholesterol and triglycerides were monitored over time after the administration of AAV-PCSK9 shRNA or AAV-GFP, n = 12 or 15 per group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (E and F) Immunoblotting results depicting hepatic levels of PCSK9, LDL-R, and ApoB-100 proteins at 6 months and 2 weeks post-administration, with corresponding quantification shown on the right, n = 5 or 6/group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01 (Unpaired t test). (G) Serum ApoB levels (μg/mL) were assessed in mice at 2 weeks and 6 months post-administration of AAV-GFP or AAV-PCSK9 shRNA, n = 8/group. Data are shown as means ± SEM. ∗ p ≤ 0.05 (Unpaired t test). (H and I) Fast protein liquid chromatography (FPLC) profiles of cholesterol and triglycerides were analyzed from pooled serum collected 2 weeks post-administration. (J and K) FPLC assessment of cholesterol and triglyceride profiles in pooled serum at 6 months post-administration.

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, shRNA, Western Blot, Fast Protein Liquid Chromatography

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: Silencing hepatic PCSK9 protects ApoE −/− mice from atherosclerosis development Twelve-week-old ApoE −/− male mice were injected with 2 × 10 11 viral genomes (v.g.) of AAV-shPCSK9 or AAV-GFP and fed a chow diet for 6 months. (A) Representative Oil Red O-stained images of the en face arterial tree. (B–F) Quantification of the lesion areas in (A), n = 8/group. (G) Representative Oil Red O staining in aortic sinus cross-sections, scale bar indicates 400 μm. (H) Quantification of the lesion areas in (G), n = 7 or 8/group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p < 0.01 (Unpaired t test).

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Injection, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: Silencing PCSK9 improves plaque stability in aortic sinus lesions (A) Representative images of macrophages and smooth muscle cells by immunostaining with CD68 antibody (red) and α-smooth muscle actin antibody (red) in aortic sinus lesions, scale bar indicates 100 μm, plaque areas circled with yellow solid lines. (B) Quantification of macrophage-positive areas in (A), n = 4 or 5/group. (C) Quantification of smooth muscle cell-positive areas in (A). n = 4 or 5/group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p < 0.01 (Unpaired t test).

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Immunostaining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE −/− mice

doi: 10.1016/j.omtm.2024.101390

Figure Lengend Snippet: Silencing PCSK9 attenuates atherosclerotic aorta and serum inflammatory monocyte infiltration and inflammatory gene expression in ApoE −/− mice (A) Representative images and quantification (B) of Ly6C hi -positive cells via aortic sinus lesions immunostaining, scale bar indicates 25 μm, n = 4 or 5/group. (C) Real-time PCR analysis of MCP-1 , TNF- α, MIF , IL-1β , IL-6 , and IL-10 mRNA expression in aortas from AAV-transduced ApoE −/− mice fed a chow diet for 6 months, n = 3 or 4/group. (D–F) Serum levels of MCP-1, MIF, and TNF-α from AAV-transduced ApoE −/− mice fed a chow diet for 6 months via ELISA, n = 7–12/group. Data are shown as means ± SEM. ∗ p ≤ 0.05, ∗∗ p < 0.01 (Unpaired t test).

Article Snippet: We measured plasma mouse PCSK9 levels using a mouse Quantikine ELISA Kit (catalog number, MPC900; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Gene Expression, Immunostaining, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial LRP1 regulates metabolic responses by acting as a co-activator of PPARγ

doi: 10.1038/ncomms14960

Figure Lengend Snippet: ( a – f ) Plasma levels of LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), TG, total cholesterol, adiponectin and leptin were analysed in Cre+ and Cre− mice before and after HFD feeding for 16 weeks (Weeks 0 or 16, respectively). ( g ) Daily food intake was monitored for both Cre+/BMT and Cre−/BMT mice individually along the whole period of HFD feeding. ( h – j ) VO 2 ( h ), locomotor activity in x axis (XTOT, i ) and z axis (ZTOT, j ) were measured in mice by metabolic cage studies before HFD feeding. ( k , l ) Plasma levels of fasting glucose and insulin were analysed in Cre+/BMT and Cre−/BMT mice before and after HFD. ( m ) Insulin tolerance tests were performed with Cre+/BMT and Cre−/BMT mice before HFD feeding (Week 0). ( n , o ) Glucose tolerance tests (GTT) were performed with Cre+/BMT and Cre−/BMT mice after HFD feeding for 16 weeks (Week 16). The area under the curve for the glucose tolerance test was quantified and presented in o . n =8 for Cre+/BMT mice and 6 for Cre−/BMT mice. * P <0.05. Analysis was two-way analysis of variance followed by Fisher's least significant difference multiple comparison test (for a – n ) and unpaired Student's t -test (for i ).

Article Snippet: The lipid contents were measured with Infinity TG and cholesterol kits (Thermo Scientific), HDL/LDL cholesterol kits (Teco Diagnostics, Anaheim, California) and fast protein liquid chromatography (FPLC) was performed by the Mouse Metabolism Core Facility (Baylor College of Medicine).

Techniques: Clinical Proteomics, Activity Assay, Comparison

Journal: Nature Communications

Article Title: Endothelial LRP1 regulates metabolic responses by acting as a co-activator of PPARγ

doi: 10.1038/ncomms14960

Figure Lengend Snippet: ( a ) Metabolic parameters including plasma levels of LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), TG, total cholesterol, adiponectin, leptin, fasting glucose and insulin were analysed in Cre+/BMT and Cre−/BMT mice after pioglitazone treatments for 4 weeks. ( b ) VO 2 , VCO 2 , locomotor activity in x axis (XTOT) and z axis (ZTOT) and RER (respiratory exchange rate) were measured in mice by metabolic cage studies after the treatment of pioglitazone. ( c ) Insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were performed with Cre+/BMT and Cre−/BMT mice after the treatment of pioglitazone. * P <0.05. n =3 for both Cre+/BMT mice and Cre−/BMT mice. Analysis was unpaired Student's t -test (for a ) and two-way analysis of variance followed by Fisher's least significant difference multiple comparison test (for b , c ).

Article Snippet: The lipid contents were measured with Infinity TG and cholesterol kits (Thermo Scientific), HDL/LDL cholesterol kits (Teco Diagnostics, Anaheim, California) and fast protein liquid chromatography (FPLC) was performed by the Mouse Metabolism Core Facility (Baylor College of Medicine).

Techniques: Clinical Proteomics, Activity Assay, Comparison

Journal: Nature Communications

Article Title: Endothelial LRP1 regulates metabolic responses by acting as a co-activator of PPARγ

doi: 10.1038/ncomms14960

Figure Lengend Snippet: ECs following different treatments were treated with 20 μg ml −1 oxLDL for determining internalized cholesterol contents. ( a ) Lrp1 shRNA or control shRNA stably transfected MECs were loaded with oxLDL for the indicated time periods. ( b ) Both isolated ECs were treated with oxLDL and 10 μM pioglitazone for 24 h. ( c ) Isolated primary MECs were transfected with the indicated plasmids. ( d ) Primary MECs were transfected with Pparγ-specific or control siRNAs and also transfected with Lrp1β or treated with pioglitazone at 10 μM or both. ( e ) Cre+ or Cre− ECs were treated with palmitic acids at 0.5 mM for 24 h. ( f ) ECs were isolated from Cre+ or Cre− mice after HFD feeding for 9 weeks. n =3. * P <0.05, compared to control shRNA or siRNA-transfected ECs or Cre− control cells. # P <0.05, compared to control shRNA transfected ECs or Cre− cells upon same treatments, except indicated comparisons in c , e , f . ** P <0.05. Analysis was two-way analysis of variance (ANOVA) followed by Fisher's least significant difference multiple comparison test (for a , b , d – f ) and one-way ANOVA followed by Bonferroni test (for c ).

Article Snippet: The lipid contents were measured with Infinity TG and cholesterol kits (Thermo Scientific), HDL/LDL cholesterol kits (Teco Diagnostics, Anaheim, California) and fast protein liquid chromatography (FPLC) was performed by the Mouse Metabolism Core Facility (Baylor College of Medicine).

Techniques: shRNA, Control, Stable Transfection, Transfection, Isolation, Comparison

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) Total serum cholesterol from overnight-fasted 12-week-old male SCoR2 +/+ (n = 20) and SCoR2 −/− mice (n = 21). (B and C) Total serum cholesterol (B) and serum triglycerides (C) from unfasted and overnight-fasted 24-week-old male SCoR2 +/+ (n = 14 for unfasted, n = 13 for fasted) and SCoR2 −/− mice (n = 14 for unfasted, n = 12 for fasted). (D) Serum from 24-week-old unfasted male SCoR2 +/+ mice (n = 7) and SCoR2 −/− mice (n = 7) were each pooled, then separated by fast protein liquid chromatography to observe lipoprotein fractions. Lipoproteins were identified compared with known standards. (E) Graphical representation of the change in LDL (top) and HDL (bottom) cholesterol fractions from pooled samples in (D). (F) Western blot analysis of PCSK9, LDLR, and HMGCR S-nitrosylation status (SNO) in livers from unfasted 24-week-old male SCoR2 +/+ and SCoR2 −/− mice. *Denotes the mature, processed form of PCSK9. Control: SNO-RAC assay performed without ascorbate. (G) Quantification (n = 4) of bands from (F). SNO-proteins were normalized to total protein for each lane and total protein was normalized to GAPDH prior to analysis. Representative SCoR2 and GAPDH blots are shown in (F). (H) Western blot analysis for hepatic LDLR and PCSK9 from unfasted 24-week-old male SCoR2 +/+ and SCoR2 −/− mice. *Denotes the mature, processed form of PCSK9. (I) Quantification (n = 7) of PCSK9 protein levels from (H) and quantitative real-time PCR analysis (n = 7) of hepatic PCSK9 mRNA from the same tissue. (J and K) Serum PCSK9 from unfasted (J) and overnight-fasted (K) 24-week-old male SCoR2 +/+ (n = 16 for unfasted, n = 13 for fasted) and SCoR2 −/− (n = 16 for unfasted, n = 12 for fasted) mice. (L) Quantification (n = 7) of LDLR protein levels from (H) and quantitative real-time PCR analysis (n = 7) of hepatic LDLR mRNA from the same tissue. (M) Total serum cholesterol from unfasted 16-week-old male SCoR2 +/+ /LDLR −/− (n = 12) and SCoR2 −/− /LDLR −/− mice (n = 7). See also and . In all figures, all bars represent mean ± SD, and all bands were quantified using ImageJ. p values in all figures were calculated by Student’s t test (unless noted otherwise).

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Fast Protein Liquid Chromatography, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) Western blot analysis of SNO-PCSK9 in HepG2 cells stably expressing control or SCoR2-targeting shRNA. *Denotes the mature, processed form of PCSK9. (B) Quantification (n = 3) of SNO-PCSK9 from (A) and related experiments. (C) Western blot analysis for cellular and secreted (media) PCSK9 in HepG2 cells stably expressing control or SCoR2-targeting shRNA. An equal number of cells were cultured for 24 h in serum-free Opti-MEM media, washed with PBS, given fresh (PCSK9-free) Opti-MEM, and harvested after 3 h. *Denotes the mature, processed form of PCSK9. (D) Quantification (n = 6) of cellular PCSK9 (left; mature PCSK9 normalized to GAPDH input) and secreted (media) PCSK9 (right; normalized to mature PCSK9 band) from (C) and related experiments. (E) Western blot analysis for LDLR in HepG2 cells stably expressing control or SCoR2-targeting shRNA. (F) Quantification (n = 3) of LDLR from (E). Total LDLR was normalized to GAPDH input. (G) Western blot analysis of SNO-PCSK9 in SCoR2-deficient HEK293 cells transiently reconstituted with SCoR2. Upper band: pro-PCSK9; lower band: mature PCSK9. (H) Quantification (n = 3) of SNO-PCSK9 from (G). (I) Western blot analysis for cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 cells transiently reconstituted with SCoR2. An equal number of cells were cultured for 24 h in serum-free Opti-MEM media, washed with PBS, given fresh (PCSK9-free) Opti-MEM, and harvested after 90 min. Upper band: pro-PCSK9; lower band: mature PCSK9. (J) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9 band) from (I). (K) Western blot analysis of SNO-SAR1A/B, SNO-SURF4, and SNO-SEC24A in SCoR2-deficient HEK293 cells with or without reconstitution of SCoR2. V5-tagged SURF4 was overexpressed in both conditions and anti-V5 antibody used to visualize SURF4. (L) Quantification (n = 3) of SNO-SAR1A/B (normalized to total SAR1A/B), SNO-SURF4 (normalized to total SURF4), and SNO-SEC24A (normalized to total SEC24A) from (K). Note SCoR2 and GAPDH immunoblots are shared between (G) and (K), as they are from the same experiment. (M) Western blot analysis of SEC23A and SAR1A/B S-nitrosylation status in livers from unfasted 24-week-old SCoR2 +/+ and SCoR2 −/− mice. (N) Quantification (n = 4) of bands from (M). SNO-protein was normalized to total protein for each lane and total protein was normalized to GAPDH prior to analysis. (O) Representative western blot analysis of SNO-SAR1A/B in HepG2 cells stably expressing control or SCoR2-targeting shRNA. (P) Quantification (n = 6) of SNO-SAR1A/B (normalized to total SAR1A/B) from (O). (Q) Western blot analysis of hepatic SEC24A and SURF4 from unfasted 24-week-old male SCoR2 +/+ and SCoR2 −/− mice. Note, SNO-SEC24A and SNO-SURF4 were assayed but no signal was detected. (R) Quantification (n = 7) of bands from (Q). Total target protein was normalized to GAPDH. In the above panels, SNO-proteins were captured from cell lysates by SNO-RAC, separated by SDS-PAGE, and analyzed by western blot. Control: SNO-RAC assay for SNO-proteins performed without ascorbate. See also .

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Cell Culture, SDS Page

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) SAR1B nitrosylates SURF4. FLAG-tagged SNO-SAR1B was incubated with V5-tagged SURF4. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. (B) SURF4 nitrosylates PCSK9. V5-tagged SNO-SURF4 was incubated with FLAG-tagged PCSK9. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. Mature PCSK9 is visualized in SNO-PCSK9 lanes. (C) Western blot analysis of SNO-PCSK9 and SNO-SAR1B in SCoR2-deficient HEK293 transfected with wild-type PCSK9 and wild-type SAR1B and treated with 200 μM ethyl ester S-nitroso-cysteine (ECySNO) for 90 min. Anti-FLAG antibody was used to visualize SAR1B. (D) Quantification (n = 3) of SNO-PCSK9 and SNO-SAR1B (normalized to total PCSK9 and SAR1B, respectively) from (C) and related experiments. (E) Western blot analysis of SNO-SAR1B wild-type and indicated mutations in SCoR2-deficient HEK293 transfected with SAR1B wild-type and indicated mutations and treated with 200 μM ECySNO for 90 min. Anti-FLAG antibody was used to visualize SAR1B in a single experiment that is verified in subsequent assays. (F) Western blot analysis of SNO-PCSK9, SNO-SURF4, and SNO-SAR1B in SCoR2-deficient HEK293 cells transiently overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (G) Quantification (n = 3) of SNO-PCSK9 (mature band, normalized to total mature PCSK9) and SNO-SURF4 from (F). (H) Representative western blot analysis for cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 cells overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (I) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9 band) from (H). p values in (I) were calculated by one-way ANOVA. (J) SCoR-deficient HEK293 cells stably expressing PCSK9 were treated with or without 200 μM ECySNO (+SNO) for 90 min then stained with anti-PCSK9 (green) and anti-calnexin (red, ER marker) antibodies. Scale bar, 5 μm. (K) Quantification of mean PCSK9 signal intensity in pixels positive for calnexin (n = 12 cells per condition). Control: SNO-RAC assay performed without ascorbate. See also .

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Incubation, Western Blot, Transfection, Stable Transfection, Expressing, Staining, Marker, Control

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) Western blot analysis of SNO-SURF4 in SCoR2-deficient HEK293 transfected with wild-type SURF4 and treated with 200μM ECySNO for 90 min. Anti-V5 antibody was used to visualize SURF4. (B) Quantification (n = 3) of SNO-SURF4 (normalized to total SURF4) from (A). (C) Western blot analysis of SNO-SURF4 in SCoR2-deficient HEK293 transfected with SURF4 wild-type or indicated mutants and treated with 200 μM ECySNO for 90 min. Anti-V5 antibody was used to visualize SURF4 in a single experiment that is verified in subsequent assays. (D) Western blot analysis of SNO-PCSK9 and SNO-SURF4 in SCoR2-deficient HEK293 cells transiently overexpressing SURF4 WT or SURF C32A and treated with 200 μM ECySNO for 90 min prior to harvest. (E) Quantification (n = 3) of SNO-PCSK9 (mature band, normalized to total mature PCSK9) from (D). (F) Western blot analysis for cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 cells overexpressing SURF4 WT or SURF4 C32A and treated with 200 μM ECySNO for 90 min prior to harvest. (G) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9 band) from (F). (H) Western blot analysis of PCSK9–SURF4 interaction with or without ECySNO treatment. An equal number of cells were transfected and cultured for 24 h, washed with PBS, given fresh (PCSK9-free) Opti-MEM media with or without 200 μM ECySNO, and harvested after 20 min. SURF4 was visualized with anti-V5 antibody. (I) Quantification (n = 3) of PCSK9–SURF4 interaction with or without ECySNO treatment from (H). (J) Western blot analysis of cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 transiently expressing PCSK9 WT or PCSK9 C301A with or without ECySNO treatment. An equal number of cells were transfected and cultured for 24 h, washed with PBS, given fresh (PCSK9-free) Opti-MEM media with or without 200 μM ECySNO, and harvested after 90 min. (K) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9, lower band) from (J) and related experiments. p values in (G) and (K) were calculated by one-way ANOVA. In the above panels, SNO-proteins were captured from cell lysates by SNO-RAC, separated by SDS-PAGE, and analyzed by western blot. Control; SNO-RAC assay for SNO-proteins performed without ascorbate. For PCSK9, upper band: pro-PCSK9; lower band: mature PCSK9. See also

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Western Blot, Transfection, Cell Culture, Expressing, SDS Page, Control

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) SNO-CoA reductase activity in liver lysate from control-fed C57BL/6J mice or mice fed diet containing AL-1576 for 4 weeks (n = 3). (B) Total serum cholesterol from 6-h fasted 20-week old male mice fed control diet (n = 10) or fed diet containing AL-1576 for 4 weeks (n = 10). (C) Pooled serum from 6-h fasted 20-week-old male mice fed control diet (n = 7) or fed diet containing AL-1576 for 4 weeks (n = 7) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Lipoprotein fractions were labeled according to known standards. (D) Serum PCSK9 from 6-h fasted 20-week-old male mice fed control diet (n = 10) or fed diet containing AL-1576 for 4 weeks (n = 10). (E) Western blot analysis of SNO-PCSK9 in livers from 6-h fasted 20-week-old male mice fed control diet (n = 4) or fed diet containing AL-1576 for 4 weeks (n = 4). Control; SNO-RAC assay for SNO-PCSK9 performed without ascorbate. Mature PCSK9 is visualized. (F) Quantification (n = 4) of bands from (E). SNO-protein was normalized to total protein for each lane and total protein was normalized to GAPDH prior to analysis. (G) Western blot analysis for hepatic LDLR and PCSK9 from 6-h fasted 20-week-old mice fed control diet or fed diet containing AL-1576 for 4 weeks. Mature PCSK9 is visualized. (H) Quantification (n = 7) of LDLR and PCSK9 protein levels from (G). (I) Quantitative real-time PCR analysis of hepatic SREBP2, LDLR, and PCSK9 from 6-h fasted 20-week-old mice fed control diet (n = 7) or fed diet containing AL-1576 for 4 weeks (n = 7). (J) Total serum cholesterol from 6-h fasted 16-week-old male LDLR −/− mice (n = 10) or LDLR −/− mice fed diet containing AL-1576 for 4 weeks (n = 9). (K) Total serum cholesterol from 6-h fasted 16-week-old male CETP/ApoB100 transgenic mice fed control diet (n = 3) or fed diet containing AL-1576 for 8 weeks (n = 4). (L) Pooled serum from 6-h fasted 16-week-old male CETP/ApoB100 transgenic mice fed control diet (n = 3) or fed diet containing AL-1576 for 8 weeks (n = 4) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Lipoprotein fractions were labeled according to known standards. (M) Total serum cholesterol from 6-h fasted 20-week-old male ApoE −/− mice fed control diet (n = 10) or fed diet containing AL-1576 for 4 weeks (n = 10). (N) Serum from 6-h fasted 20-week-old male ApoE −/− mice fed control diet (n = 10) or diet containing AL-1576 for 4 weeks (n = 10) was separated by fast protein liquid chromatography to obtain individual lipoprotein fractions. Lipoprotein fractions were labeled according to known standards. (O) Serum PCSK9 from 6-h fasted 20-week-old male ApoE −/− mice fed control diet (n = 10) or diet containing AL-1576 for 4 weeks (n = 10). (P) Western blot analysis for hepatic LDLR and PCSK9 from 6-h fasted 20-week-old ApoE −/− mice fed control diet or diet containing AL-1576 for 4 weeks. (Q) Quantification (n = 7) of LDLR and PCSK9 protein levels from (P).

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Activity Assay, Control, Fast Protein Liquid Chromatography, Labeling, Western Blot, Real-time Polymerase Chain Reaction, Transgenic Assay

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (Left) SCoR2 enables PCSK9 secretion by preventing S-nitrosylation of COPII components (SAR1, and SURF4) and cargo (PCSK9). PCSK9 can bind its cargo receptor SURF4 to promote selection of PCSK9 into nascent ER vesicles. (Right) SCoR2 inhibition (genetically or pharmacologically) blocks PCSK9 secretion via an S-nitrosylation cascade thereby lowering LDL cholesterol. Specifically, inhibition of SCoR2 leads to increases in SAR1 S-nitrosylation; SNO-SAR1 then acts as a nitrosylase for SURF4 to form SNO-SURF4, which then nitrosylates PCSK9 to inhibit PCSK9-SURF4 interaction. That is, SNO-PCSK9 binding to SURF4 is ineffectual, preventing selection of PCSK9 into nascent ER vesicles, thereby reducing PCSK9 secretion. Created with BioRender.com .

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Selection, Inhibition, Binding Assay

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet:

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Recombinant, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, shRNA, Mutagenesis, Software