Journal: PLOS Pathogens

Article Title: Nicotinamide metabolism is essential for Hepatitis C Virus replication and the production of infectious Lipo-Viro-Particles

doi: 10.1371/journal.ppat.1014165

Figure Lengend Snippet: Huh7 cells were treated or not with 100 µM 6-AN for 72h of culture. (A-B) ApoB and ApoE secreted in cells supernatant quantified by ELISA. Are presented means ± SEM (n = 3, student’s t-test, **p < 0.0021, ***p < 0.0002). (C) Quantification of intracellular triglycerides in total cell extracts. Are presented means ± SEM (n = 3, student’s t-test, *p < 0.01). (D) Representative picture of 3D lipid droplets reconstruction using IMARIS software in Huh7 cells after Oil-red-O (yellow) and Hoechst (blue) staining. (E) The mean total volume of lipid droplets per cell was determined by analyzing their intracellular content of 50 cells for each condition with the IMARIS software. Are presented means ± SEM, one-way ANOVA for multiple comparison, n.s. non significative, ****p < 0.0001.

Article Snippet: ApoB and ApoE concentrations in medium and gradients fractions were determined by ELISA as previously described [ ], using the following antibodies: anti-human ApoB mAbs (LDL 20/17), anti-human ApoB mAb (LDL 11) biotin, anti-human ApoE mAb (E276), anti-human ApoE mAb (E887) biotin (MABTECH, Sweden).

Techniques: Enzyme-linked Immunosorbent Assay, Software, Staining, Comparison

Journal: Cancer Management and Research

Article Title: Apolipoprotein E (APOE) Promotes Cell Proliferation and Invasion in Glioma via the PI3K/AKT Signaling Pathway

doi: 10.2147/CMAR.S594906

Figure Lengend Snippet: Nuclear translocation of APOE activates the PI3K/AKT signaling pathway in glioma cells. ( A ) Immunofluorescence staining showing the subcellular localization of APOE in U251 cells. ( B ) Western blot analysis of APOE protein in nuclear and cytoplasmic fractions of U251 cells. ( C ) Coomassie Brilliant Blue staining of immunoprecipitated APOE protein complexes in U251 cells. ( D ) Volcano plot showing differentially expressed genes (DEGs) in APOE-overexpressing U251 cells compared with control cells. A total of 443 DEGs were identified. ( E ) Gene Ontology (GO) enrichment analysis of the DEGs. ( F ) KEGG pathway enrichment analysis of the DEGs. ( G ) Western blot analysis and quantification of p-PI3K/PI3K and p-AKT/AKT ratios in APOE-overexpressing U251 cells treated with LY294002. Protein phosphorylation levels were quantified and analyzed using unpaired two-tailed t tests with false discovery rate (FDR) correction. Differences were considered statistically significant at q < 0.05. ( H ) Western blot analysis and quantification of p-PI3K/PI3K and p-AKT/AKT ratios in APOE-overexpressing U87 cells treated with LY294002. Protein phosphorylation levels were quantified and analyzed using unpaired two-tailed t tests with false discovery rate (FDR) correction. Differences were considered statistically significant at q < 0.05. ( I ) Western blot analysis and quantification of p-PI3K/PI3K and p-AKT/AKT ratios in APOE knockout U251 cells. Protein phosphorylation levels were quantified and analyzed using unpaired two-tailed t tests with false discovery rate (FDR) correction. Differences were considered statistically significant at q < 0.05. (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The membrane was subsequently blocked with 5% nonfat dry milk for 30‒60 min and then incubated with the following primary antibodies overnight at 4 °C: APOE (#68587, CST), PI3 kinase p85 (#660225-1-Ig, ProteinTech), phospho-PIK3R1 (p-PI3K) (#CSB- PA000712 , CUSABIO), AKT (#2920S, Cell Signaling), P-AKT (S473) (#9271s, Cell Signaling), β-actin (#66009-1-Ig, ProteinTech), Lamin B1 (#12987-1-AP, ProteinTech), and α-Tubulin (#66031-1-Ig, ProteinTech) Next, the membrane was incubated with HRP-conjugated secondary antibodies for 60 min at ambient temperature Protein band signals were captured via the Tanon 5200 system (Tanon Technology Co., Ltd.) and analyzed with ImageJ software (version 1.40 g; National Institutes of Health).

Techniques: Translocation Assay, Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Control, Phospho-proteomics, Two Tailed Test, Knock-Out

Journal: Cancer Management and Research

Article Title: Apolipoprotein E (APOE) Promotes Cell Proliferation and Invasion in Glioma via the PI3K/AKT Signaling Pathway

doi: 10.2147/CMAR.S594906

Figure Lengend Snippet: PI3K inhibition attenuates APOE-mediated proliferation and motility in glioma cells. ( A ) Cell viability of U251 cells stably overexpressing APOE or vector control following treatment with the PI3K inhibitor LY294002, assessed using the CCK-8 assay over a 4-day period, with OD450 measured daily. Data are presented as mean ± SD (n=3). Statistical significance between control and APOE-overexpressing cells across the entire observation period was determined using two-way ANOVA, with time and genotype as factors. ( B ) Transwell assays evaluating the migratory and invasive capacities of U251 cells with or without APOE overexpression and LY294002 treatment, with quantitative analysis. Representative images and quantification of migrated and invaded cells are shown. Migration and invasion were quantified by counting cells that traversed the membrane and are presented as mean ± SEM from independent experiments (n=4). Statistical analysis was performed using unpaired two-tailed t tests with false discovery rate (FDR) correction for multiple comparisons. ( C ) Wound healing assays assessing cell migration in U251 cells under the same experimental conditions as in (B), with quantitative analysis of relative wound closure area. Representative images and quantification are shown. Data are presented as mean ± SEM from independent experiments (n=3). Statistical significance was determined using unpaired two-tailed t tests. ( D ) Cell viability of U87 cells assessed by the CCK-8 assay following PI3K inhibition. Data are presented as mean ± SD (n=3). Statistical significance between control and APOE-overexpression cells across the entire observation period was determined using two-way ANOVA, with time and genotype as factors. ( E ) Transwell migration and invasion assays of U87 cells following PI3K inhibition, with quantitative analysis. Representative images and quantification of migrated and invaded cells are shown. Migration and invasion were quantified by counting cells that traversed the membrane and are presented as mean ± SEM from independent experiments (n=4). Statistical analysis was performed using unpaired two-tailed t tests with false discovery rate (FDR) correction for multiple comparisons. (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membrane was subsequently blocked with 5% nonfat dry milk for 30‒60 min and then incubated with the following primary antibodies overnight at 4 °C: APOE (#68587, CST), PI3 kinase p85 (#660225-1-Ig, ProteinTech), phospho-PIK3R1 (p-PI3K) (#CSB- PA000712 , CUSABIO), AKT (#2920S, Cell Signaling), P-AKT (S473) (#9271s, Cell Signaling), β-actin (#66009-1-Ig, ProteinTech), Lamin B1 (#12987-1-AP, ProteinTech), and α-Tubulin (#66031-1-Ig, ProteinTech) Next, the membrane was incubated with HRP-conjugated secondary antibodies for 60 min at ambient temperature Protein band signals were captured via the Tanon 5200 system (Tanon Technology Co., Ltd.) and analyzed with ImageJ software (version 1.40 g; National Institutes of Health).

Techniques: Inhibition, Stable Transfection, Plasmid Preparation, Control, CCK-8 Assay, Over Expression, Migration, Membrane, Two Tailed Test

Journal: Cancer Management and Research

Article Title: Apolipoprotein E (APOE) Promotes Cell Proliferation and Invasion in Glioma via the PI3K/AKT Signaling Pathway

doi: 10.2147/CMAR.S594906

Figure Lengend Snippet: APOE promotes glioma growth in vivo through activation of the PI3K/AKT pathway. ( A ) Representative images of subcutaneous tumors formed in nude mice (n = 3 per group). ( B ) Tumor growth curves of U251 xenografts over a 56-day period. Tumor volumes were measured at the indicated time points in mice bearing U251 xenografts with APOE knockout (KO), control, APOE overexpression (APOE OE), or APOE OE treated with the PI3K inhibitor LY294002. Data are presented as mean ± SEM (n=3). Statistical significance was determined using two-way ANOVA, with time and treatment as factors. ( C ) Statistical comparison of tumor weights at the end of the 56-day experimental period. Data are presented as mean ± SEM (n=3). Statistical significance was determined using an unpaired two-tailed t test. ( D ) Body weight curves of nude mice over the 56-day experimental period. Data are presented as mean ± SEM (n=3). Statistical significance was determined using two-way ANOVA, with time and treatment as factors. ( E ) Immunohistochemical (IHC) staining of APOE, PI3K, p-PI3K, AKT, and p-AKT in subcutaneous tumor tissues. ( F ) Western blot analysis and quantification of APOE, PI3K, p-PI3K, AKT, and p-AKT expression levels in subcutaneous tumors from nude mice. APOE and protein phosphorylation levels were quantified and analyzed using unpaired two-tailed t tests with false discovery rate (FDR) correction. Differences were considered statistically significant at q < 0.05. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membrane was subsequently blocked with 5% nonfat dry milk for 30‒60 min and then incubated with the following primary antibodies overnight at 4 °C: APOE (#68587, CST), PI3 kinase p85 (#660225-1-Ig, ProteinTech), phospho-PIK3R1 (p-PI3K) (#CSB- PA000712 , CUSABIO), AKT (#2920S, Cell Signaling), P-AKT (S473) (#9271s, Cell Signaling), β-actin (#66009-1-Ig, ProteinTech), Lamin B1 (#12987-1-AP, ProteinTech), and α-Tubulin (#66031-1-Ig, ProteinTech) Next, the membrane was incubated with HRP-conjugated secondary antibodies for 60 min at ambient temperature Protein band signals were captured via the Tanon 5200 system (Tanon Technology Co., Ltd.) and analyzed with ImageJ software (version 1.40 g; National Institutes of Health).

Techniques: In Vivo, Activation Assay, Knock-Out, Control, Over Expression, Comparison, Two Tailed Test, Immunohistochemical staining, Immunohistochemistry, Western Blot, Expressing, Phospho-proteomics

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Preprocessing of single-cell RNA sequencing (scRNA-seq) dataset and identification of synovial cell in GSE192504 database. ( A and B ) Highly variable genes filtering and PCA clustering of scRNA-seq data on the synovial cells isolated from the collagen-induced arthritis (CIA) mice group and the healthy control mice (normal) group. ( C and D ) Annotations and the proportions of different cell types between the CIA and normal group. Monocytes, macrophages, and fibroblasts accounted for significant proportions of synovial cells in the CIA group compared to the normal group. ( E ) t-SNE projections and cell annotation of scRNA-seq data extracted from the CIA group. Synovial cells mainly included monocytes, macrophages, and fibroblast-like synoviocytes in the CIA group. Cell types were annotated based on canonical marker gene expression (see Methods). ( F ) The expression levels and distribution of 10 hub genes in different synovial cells of the CIA group. Violin plots and t-SNE plots both showed specifical high expression of APOE and CCR2 in macrophages.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Single Cell, RNA Sequencing, Isolation, Control, Marker, Gene Expression, Expressing

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Correlation analysis between two hub gene expressions and GSVA immune-enrichment scores. ( A and B ) The heatmaps showed APOE and CCR2 expression and functional enrichment scores of RA patients in the GSE48780 database. The Spearman R and P values were respectively visualized by using the right bar graphs and line graphs.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Expressing, Functional Assay

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Changes in each macrophage cluster upon stimulation with LPS/IFN-γ or IL-4. ( A ) Representative images of immunofluorescence of each macrophage cluster. Blue represented the genetic material stained with DAPI and green showed fluorescence of APOE, PDPN, CD74, and PLAC8, which are regarded as specific protein markers of macrophage clusters. ( B ) Fluorescence intensity of APOE, PDPN, CD74, and PLAC8. Data are shown as mean ± SD from three independent biological experiments. * P < 0.05; *** P < 0.001.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Inflammation Research

Article Title: APOE and CCR2 : Potential Macrophage-Specific Biomarkers in the Rheumatoid Arthritis Synovial Microenvironment Identified by Bioinformatics and Experimental Verification in Murine Models

doi: 10.2147/JIR.S587712

Figure Lengend Snippet: Gene and protein expression levels of APOE and CCR2 in LPS/IFN-γ or IL-4-stimulated RAW 264.7 cells. ( A ) Relative gene expression levels of APOE and CCR2 at 12h and 24 h after the addition of LPS/IFN-γ or IL-4. ( B ) APOE and CCR2 protein levels analyzed by ELISA at 24h upon stimulation with LPS/IFN-γ or IL-4. Data are shown as mean ± SD from three independent biological experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: The sections were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation at 4 °C with the following primary antibodies (1:100 dilution): anti-apolipoprotein E (APOE) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-placenta specific 8 (PLAC8) (rabbit monoclonal antibody, Proteintech Group, Inc., China), anti-CD74 (mouse monoclonal antibody, Proteintech Group, Inc., China), anti-podoplanin (PDPN) (mouse monoclonal antibody, Proteintech Group, Inc., China).

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay