Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Volcano plot of differentially expressed genes from RNA sequencing, showing upregulation of PCSK9 and downregulation of APOA4 in the LD+TMAO group compared to the LD group. (B) qRT-qPCR analysis of APOA4 mRNA expression levels across groups (n = 3 per group). (C) qRT-qPCR analysis of PCSK9 mRNA levels across groups (n = 3 per group). (D) Western blot analysis of APOA4 and PCSK9 protein levels across groups (n = 3 per group). (E–F). Quantification results of the Western blot analysis using ImageJ. (G, H). Serum APOA4 and PCSK9 levels in patients with cholelithiasis and the control group detected by ELISA. ** p < 0.01, *** p < 0.001. LD, lithogenic diet; TMAO, trimethylamine-N-oxide; NC, negative control; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; GSD, gallstone disease; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) Lithogenesis in mice from the LD and LD+DMB groups. (B) Weights of solid contents in the gallbladders of mice from each group. (C) Serum TMAO levels in the LD and LD+DMB groups detected by ELISA. (D) Immunofluorescence staining of APOA4 and PCSK9 in hepatic tissues from the LD and LD+DMB groups. (E, F) qRT-qPCR analysis of APOA4 and PCSK9 mRNA expression levels in hepatic tissues from the LD and LD+DMB groups. * p < 0.05, ** p < 0.01, **** p < 0.0001. LD, lithogenic diet; DMB, 3,3-dimethyl-1-butanol; TMAO, trimethylamine-N-oxide; ELISA, enzyme-linked immunosorbent assay; APOA4, apolipoprotein A4; PCSK9 proprotein convertase subtilisin/kexin type 9.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A–E) APOA4 , PCSK9 , HMGCR, ABCG5 , and ABCG8 mRNA expression levels in TMAO-treated and control AML12 cells (n = 3 per group). (F) Immunofluorescence staining for APOA4, PCSK9, HMGCR, ABCG5, and ABCG8 in cells from each group. ** p < 0.01, *** p < 0.001. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Expressing, Control, Immunofluorescence, Staining, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A) WB analysis of PCSK9, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and APOA4-overexpressed cells. (B–E) Changes in PCSK9 , HMGCR , ABCG5 , and ABCG8 mRNA levels after APOA4 overexpression (n = 3 per group). (F) Immunofluorescence staining for intracellular PCSK9, HMGCR, ABCG5, and ABCG8 after APOA4 overexpression. (G) WB analysis of APOA4, HMGCR, ABCG5, and ABCG8 in NC, TMAO-treated, and PCSK9-knockdown cells. (H–K) Changes in mRNA levels of PCSK9 , HMGCR , ABCG5 , and ABCG8 after PCSK9 knockdown (n = 3 per group). (L) Immunofluorescence staining for PCSK9, HMGCR, ABCG5, and ABCG8 in cells after PCSK9 knockdown. * p < 0.05, ** p < 0.01. +, positive expression; -, negative expression; TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Over Expression, Immunofluorescence, Staining, Knockdown, Expressing, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: Gut microbiota produce trimethylamine, which enters the liver via the portal circulation and is primarily oxidized by FMO3 to produce TMAO. TMAO upregulates hepatic PCSK9 gene expression while downregulating APOA4 expression. PCSK9 overexpression inhibits APOA4 expression, while low APOA4 expression further promotes PCSK9 expression, forming a feedback loop that dysregulates cholesterol metabolism. This upregulates cholesterol synthesis by HMGCR and cholesterol efflux by ABCG5/8. Consequently, biliary concentrations of cholesterol and bile acids increase and decrease, respectively, thereby promoting cholesterol gallstone formation. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; FMO3, flavin containing monooxygenase 3; TMA, Trimethylamine.

Article Snippet: Paraffin-embedded tissue sections were incubated overnight at 4°C with antibodies against APOA4 (Proteintech, China), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (CUSABIO, China), ATP-binding cassette sub-family G member 5 (ABCG5) (Abmart, China), PCSK9, and ATP-binding cassette sub-family G member 8 (ABCG8) (Abclonal, China).

Techniques: Gene Expression, Expressing, Over Expression, Binding Assay

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: PCR primers and conditions

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques:

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: Alb and Apoa4 expression levels in spinal cord tissues detected by qRT-PCR. (A) Venn diagram showing the intersection of two genes obtained from the WT vs SOD1 G93A + PBS group and SOD1 G93A + PBS vs SOD1 G93A + simvastatin group; (B and C) The expression levels of Apoa4 (B) and Alb (C) decreased in the SOD1 G93A + PBS group compared with the WT group but increased in the SOD1 G93A + simvastatin group compared with the SOD1 G93A + PBS group. * P < 0.05, ** P < 0.01, vs WT group; ## P < 0.01, vs SOD1 G93A + PBS group. SOD1: Superoxide dismutase 1; WT: Wilt-type; qRT-PCR: Quantitative real-time polymerase chain reaction.

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: PPI network and enrichment analysis. (A) PPI network constructed for Alb; (B) GO analysis revealed that Alb participated in lipid localization, vesicle lumen, and peptidase regulator activity; (C) KEGG pathway annotation revealed that Alb was mainly enriched in complement and coagulation cascades and cholesterol metabolism; (D) PPI network constructed for Apoa4; (E) GO analysis revealed that Apoa4 participated in the regulation of plasma lipoprotein particle levels, protein–lipid complexes, and protein–lipid complex binding; (F) KEGG pathway annotation revealed that Apoa4 was mainly enriched in cholesterol metabolism, PPAR signaling pathway, fat digestion and absorption, and complement and coagulation cascades. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: Protein–protein interaction; PPAR: Peroxisome proliferator-activated receptor.

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques: Construct, Activity Assay, Coagulation, Clinical Proteomics, Binding Assay

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: Expression of mRNA and proteins. qRT-PCR results presented the expression levels of PI3K (A), AKT (B), C5 (C), PPARγ (D), C4a (E), C3 (F), C1qB (G), and CRP (H) mRNA in different groups; the expression levels of PI3K, p-PI3K, p-AKT, AKT, PPARγ, Apoa4 (I), and C4a, C5, C3, Alb, C1qB, and CRP (J) in different groups were detected. The levels of p-PI3K, p-AKT, PPARγ, Apoa4, C5, and Alb decreased, while C4a, C3, C1qB, and CRP increased in the SOD1G93A + PBS group compared with the WT group; * P < 0.05, ** P < 0.01, vs WT group; # P < 0.05, ## P < 0.01, vs SOD1 G93A + PBS group. qRT-PCR: Quantitative real-time polymerase chain reaction; SOD1: Superoxide dismutase 1; WT: Wilt-type; PPAR: Peroxisome proliferator-activated receptor.

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Molecular Biosciences

Article Title: Serum proteomic profiling of precancerous gastric lesions and early gastric cancer reveals signatures associated with systemic inflammatory response and metaplastic differentiation

doi: 10.3389/fmolb.2024.1252058

Figure Lengend Snippet: DEPs coincident with the tissue expression. (A) Numbers of DEPs coincident with the tissue expression. (B) DEPs which were mainly expressed by enterocytes in single-cell RNA data of gastric tissue samples. (C) DEPs with high expression specificity in enterocyte in Human Protein Atlas data. (D) APOA4 levels in the gastric tissue samples. The barplot is the statistical result of the optical density analysis of IHC images. These tissue samples include NAG ( n = 10), IM ( n = 16), and GC ( n = 16). * p < 0.05; ** p < 0.01. (E) DEPs which were mainly expressed by epithelial cells in single-cell RNA data. (F) Fold changes of DEPs across cell types in pre-GC vs gastritis/normal comparison group. (G) DEPs coincident with the RNA datasets, GSE55696 and GSE130823. (H–J) ROC curves of the random forest models using the signature, APOA4, SERPINA4, and GCA.

Article Snippet: APOA4 antibody (17996-1-AP, Proteintech, USA) and COMP antibody (28369-1-AP, Proteintech, USA) were diluted at ratio 1:200.

Techniques: Expressing, Comparison