Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: SERPINE2 depletion alters GSRCC cell phenotypes, HUVEC tube formation and M2 macrophage polarization. (A) Expression of SERPINE2 protein in NUGC-4 and SNU-668 cells transfected with sh-SERPINE2#1, sh-SERPINE2#2 or sh-NC. (B-G) NUGC-4 and SNU-668 cells were introduced with sh-SERPINE2#1 or sh-NC. (B) In vitro cell apoptosis (flow cytometry) of transfected NUGC-4 and SNU-668 cells. (C) In vitro cell proliferation (EdU incorporation assay) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (D,E) Cell invasive and migratory capacities (Transwell assay with crystal violet staining) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (F) HUVECs were incubated with the CM from transfected NUGC-4 and SNU-668 cells and checked for tube formation ability. Scale bars: 100 µm. (G) THP-1/M0 cells were incubated with the CM from transfected NUGC-4 and SNU-668 cells and checked for the percent of the CD206 + macrophages (flow cytometry). GSRCC, gastric signet ring cell carcinoma; SERPINE2, serpin family E member 2; HUVECs, human umbilical vein endothelial cells; CM, conditioned medium; EdU, 5-ethynyl-2'-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: To induce differentiation to macrophages (THP-1/M0), THP-1 monocytes were maintained in complete media containing 100 ng/mL phytohaemagglutinin (PHA, Selleck, Shanghai, China) for 24 h. The medium was replaced with fresh medium every 2 days, and the cells were passed every 4–5 days.

Techniques: Expressing, Transfection, In Vitro, Flow Cytometry, Transwell Assay, Staining, Incubation

Journal: Translational Cancer Research

Article Title: METTL3 stabilizes SERPINE2 via the m6A modification to drive the malignant progression of gastric signet ring cell carcinoma

doi: 10.21037/tcr-24-896

Figure Lengend Snippet: METTL3 depletion alters cell phenotypes via reducing SERPINE2 expression. (A-G) NUGC-4 and SNU-668 GSRCC cells were subjected to transfection of sh-NC, sh-METTL3, or sh-METTL3 along with a SERPINE2 expression plasmid. (A) SERPINE2 protein expression in transfected NUGC-4 and SNU-668 cells. (B) Cell apoptosis (flow cytometry) of transfected NUGC-4 and SNU-668 cells. (C) Cell proliferation (EdU assay) of transfected NUGC-4 and SNU-668 cells. (D,E) Cell invasive and migratory capacities (Transwell assay with crystal violet staining) of transfected NUGC-4 and SNU-668 cells. Scale bars: 100 µm. (F) HUVECs were incubated with the CM of transfected NUGC-4 and SNU-668 cells and checked for tube formation ability. (G,H) THP-1/M0 cells were incubated with the CM of transfected NUGC-4 and SNU-668 cells and checked for the percent of the CD206 + macrophages (flow cytometry) and representative images. SERPINE2, serpin family E member 2; METTL3, methyltransferase-like 3; GSRCC, gastric signet ring cell carcinoma; EdU, 5-ethynyl-2'-deoxyuridine; HUVECs, human umbilical vein endothelial cells; CM, conditioned medium.

Article Snippet: To induce differentiation to macrophages (THP-1/M0), THP-1 monocytes were maintained in complete media containing 100 ng/mL phytohaemagglutinin (PHA, Selleck, Shanghai, China) for 24 h. The medium was replaced with fresh medium every 2 days, and the cells were passed every 4–5 days.

Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, EdU Assay, Transwell Assay, Staining, Incubation

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 mRNA expression levels in PBMCs and liver tissues from control (n = 6 per group) and ACLF (n = 6 per group) mice. (B) mRNA expression of CD86 and PDK4 in PMs isolated from control (n = 6 per group) and ACLF (n = 6 per group) mice. (C-F) CD86 and PDK4 expression in THP-1 macrophages and RAW264.7 cells after induction of M1 polarization (n = 3). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4, pyruvate dehydrogenase kinase 4; ACLF, acute-on-chronic liver failure; PBMCs, peripheral blood mononuclear cells; PMs, peritoneal macrophages; ns, not significant.

Article Snippet: THP-1 cells were differentiated into THP-1 macrophages using phorbol 12-myristate 13-acetate (PMA, MCE, NJ, USA).

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 expression was assessed by Western blot in THP-1 macrophages transfected with PDK4 overexpression plasmid (OE-PDK4) or empty vector (OE-EV). (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ** P < 0.01, *** P < 0.001, **** P < 0.0001. OE-PDK4, PDK4 overexpression plasmid; OE-EV, overexpression empty vector; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha.

Article Snippet: THP-1 cells were differentiated into THP-1 macrophages using phorbol 12-myristate 13-acetate (PMA, MCE, NJ, USA).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Cell viability of THP-1 macrophages treated with increasing concentrations of PDK4-IN (0–40 µM) for 24 h was assessed by CCK-8 assay. (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. Quantification of fluorescence intensity is shown on the right. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. (G) Flow cytometric analysis of apoptosis in LO2 cells cultured with conditioned media derived from macrophages. (H) CCK-8 assay of viability in LO2 cells cultured with conditioned media derived from macrophages. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4-IN, pyruvate dehydrogenase kinase 4 inhibitor; CCK-8, Cell Counting Kit-8; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

Article Snippet: THP-1 cells were differentiated into THP-1 macrophages using phorbol 12-myristate 13-acetate (PMA, MCE, NJ, USA).

Techniques: CCK-8 Assay, Western Blot, Flow Cytometry, Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Two Tailed Test, Cell Counting

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Single‐cell RNA sequencing analysis of aortic immune and stromal cells in ApoE −/− mice with AngII infusion and SOST ki .ApoE −/− mice with AngII infusion. (A) UMAP plot showing cell clusters identified by the Leiden algorithm, labeled 0 to 12, representing diverse cellular populations in aortic tissue. (B) Cell type annotation based on canonical markers, identifying major populations including macrophages, fibroblasts, endothelial cells, smooth muscle cells, T cells, B cells, and Schwann cells (left), as well as subdividing major populations into specific subtypes such as M1‐like pro‐inflammatory and M2‐like anti‐inflammatory macrophages, multiple endothelial subtypes, and multiple fibroblast subtypes (right). (C) Dot plot showing expression of selected marker gene across identified clusters. Dot size represented the percentage of cells in each cluster expressing the gene, while color intensity indicated the mean expression level. (D) UMAP plots showing the expression of genes including S100a8, Trem2, S100a4, Mrc1, and Rpl37 across macrophage clusters. (E) Violin plots showing expression distribution of marker genes (S100a8, Trem2, S100a4, Mrc1, and Rpl37) across macrophage clusters based on Leiden clustering. (F) UMAP plot of macrophage subtypes M1‐like 1, M1‐like 2, M2‐like, Res‐like 1, and Res‐like 2, based on manual annotation. Note: Clusters 0, 1, 2, 3, and 4 represented the following macrophage sub‐types: Cluster 0: M1‐like 1, Cluster 1: M1‐like 2, Cluster 2: M2‐like, Cluster 3: Res‐like 1, and Cluster 4: Res‐like 2. (G) Dot plot summarizing the expression of key marker genes (S100a8, Trem2, S100a4, Mrc1, and Rpl37) across macrophage subtypes. Dot size represented the fraction of cells expressing each gene, while color intensity indicated mean expression. (H) Dot plot of the TOP 5 highly expressed genes across macrophage subtypes. Dot size indicated the fraction of cells expressing each gene, and color intensity represented mean expression.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Single Cell, RNA Sequencing, Labeling, Expressing, Marker

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Comparative analysis of distribution patterns, subtype proportions, and differentiation trajectories of macrophages between ApoE −/− mice with AngII infusion and SOST ki .ApoE −/− mice with AngII infusion. (A) UMAP plot comparing differences in transcriptional profiles and spatial distribution of aortic macrophage subtypes between ApoE −/− mice (prune) and SOST ki .ApoE −/− mice (yellow). (B) Density map showing the distribution of macrophages from aortas in ApoE −/− mice. Higher cell density was indicated by red areas. (C) Density map showing the distribution of macrophages from aortas in SOST ki .ApoE −/− mice. Higher cell density was indicated by red areas. (D) Stacked bar plot comparing the difference in proportion of macrophage subtypes between ApoE −/− mice and SOST ki .ApoE −/− mice. (E) PAGA Slingshot graph for aortic macrophages in ApoE −/− mice, indicating differentiation trajectories toward pro‐inflammatory macrophage subtypes. (F) PAGA Slingshot graph for aortic macrophages in SOST ki .ApoE −/− mice, indicating differentiation trajectories toward anti‐inflammatory macrophage subtypes. Note: Clusters 0, 1, 2, 3, and 4 represented the following macrophage sub‐types: Cluster 0: M1‐like 1, Cluster 1: M1‐like 2, Cluster 2: M2‐like, Cluster 3: Res‐like 1, and Cluster 4: Res‐like 2.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques:

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Analysis of differential gene expression and functional pathway enrichment in macrophages between ApoE −/− mice with AngII infusion and SOST ki .ApoE −/− mice with AngII infusion. (A) Volcano plot showing differentially expressed genes (DEGs) in macrophages from SOST ki .ApoE −/− mice, compared to ApoE −/− mice. Red points represented significantly upregulated genes (116 genes) in SOST ki .ApoE −/− mice, blue points represented significantly downregulated genes (245 genes) in SOST ki .ApoE −/− mice. (B) Stacking violin plot showing expression distribution of marker genes across macrophage subtypes in ApoE −/− mice and SOST ki .ApoE −/− mice. (C) TOP 10 KEGG pathways enriched for differentially expressed genes across all macrophages between ApoE −/− mice and SOST ki .ApoE −/− mice.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Gene Expression, Functional Assay, Expressing, Marker

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: The suppressive effects of sclerostin on inflammatory responses were dependent on ApoER2 in both human macrophages and mouse macrophages in vitro. (A) The mRNA expression of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in human differentiated macrophages (THP‐1) with/without LPS (100 ng/mL) induction (n = 3 per group). (B) The medium protein levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in human differentiated macrophages (THP‐1) with/without LPS (100 ng/mL) induction (n = 3 per group). (C) The mRNA expression of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in mouse macrophages (RAW 264.7) with/without LPS (1 µg/mL) induction (n = 3 per group). (D) The medium protein levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in mouse macrophages (RAW 264.7) with/without LPS (1 µg/mL) induction (n = 3 per group). Data were expressed as mean ± standard deviation. One‐way ANOVA with Tukey's post‐hoc test versus siNC+LPS group was used to determine the intergroup differences. All tests were two‐sided. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 . Note: SOST: sclerostin; si Lrp8 : small interfering RNA targeting ApoER2 (encoded by Lrp8 ); siNC: negative control small interfering RNA; LPS: lipopolysaccharide; TNF‐α: tumor necrosis factor alpha; MCP‐1: monocyte chemoattractant protein‐1; IL‐10: interleukin‐10.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: In Vitro, Expressing, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Determination of the inflammatory cytokines/chemokines expression, macrophage phenotypes conversion, NF‐κB nuclear translocation/phosphorylation/expression in macrophages in vitro. (A) The mRNA expression levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 18 h (n = 3 per group). (B) The medium protein levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 18 h (n = 3 per group). (C) Surface markers examined by flow cytometry depict expression of CD206 and CD11b in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 18 h (left). Quantification of the proportion of anti‐inflammatory phenotypes (CD206 + ) (right) (n = 2 per group). (D) Representative images (left) and quantitative analysis of immunofluorescence (right) of NF‐κB (p65) nuclear translocation in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 15 min. Scale bar: 50 µm. The white arrows indicated the p65‐positive nuclei (n = 3 per group). (E) The protein level of phospho‐NF‐κB (p‐p65) in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 6 h (left). Quantification of the density of detected bands (right) (n = 2 per group). (F) The mRNA expression of NF‐κB (p65) in mouse macrophages (RAW264.7) with LPS (1 µg/mL) induction for 6 h (n = 3 per group). Data were expressed as mean ± standard deviation. One‐way ANOVA with Tukey's post‐hoc test versus LPS group was used to determine the intergroup differences. All tests were two‐sided. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 . Note: SOST: sclerostin; Lrp8 : low‐density lipoprotein receptor‐related protein 8 (encoding ApoER2); LPS: lipopolysaccharide; TNF‐α: tumor necrosis factor alpha; MCP‐1: monocyte chemoatractant protein‐1; IL‐10: interleukin‐10. CD11b: alpha chain of the macrophage‐1 receptor (macrophage marker); CD206: cluster of differentiation 206, known as mannose receptor C‐type 1 (anti‐inflammatory macrophage biomarker).

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Expressing, Translocation Assay, Phospho-proteomics, In Vitro, Flow Cytometry, Immunofluorescence, Standard Deviation, Marker, Biomarker Discovery

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Blockade of sclerostin loop2‐ApoER2 interaction by both Lrp8m and ApoER2‐Pep attenuated the suppressive effects of sclerostin on inflammatory responses in macrophages in vitro. (A) Binding analysis for the interaction of ApoER2 to full‐length sclerostin and sclerostin loop2 in macrophages (RAW264.7) by pull‐down assay. (B) The binding affinity of ApoER2 to sclerostin (upper) and sclerostin loop2 (lower), determined by biolayer interferometry (BLI) analysis. (C) Confocal microscopy examination of cell surface binding of sclerostin to ApoER2 in macrophages (RAW264.7), scale bar: 5 µm. (D) Binding analysis for the interaction of ApoER2 muteins to sclerostin loop2 by pull‐down assay. (E) Binding analysis for the interaction of ApoER2 to wild‐type SOST (SOST) and SOST loop2m in macrophages (RAW264.7) by co‐immunoprecipitation assay. (F) The binding affinity of ApoER2 to sclerostin with the pretreatment of ApoER2‐Pep peptide tool by BLI analysis. (G) The influence of Lrp8m in the effects of sclerostin on mRNA expression of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in RAW264.7 with LPS (1 µg/mL) induction for 18 h (n = 3 per group). (H) The influence of ApoER2‐Pep in the effects of sclerostin on mRNA expression of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in RAW264.7 with LPS (1 µg/mL) induction for 18 h (n = 3 per group). (I) The influence of Lrp8m in the effects of sclerostin on the protein levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in RAW264.7 with LPS (1 µg/mL) induction for 18 h (n = 3 per group). (J) The influence of ApoER2‐Pep in the effects of sclerostin on the protein levels of TNF‐α (left), MCP‐1 (middle), and IL‐10 (right) in RAW264.7 with LPS (1 µg/mL) induction for 18 h (n = 3 per group). Data were expressed as mean ± standard deviation. The unpaired t‐test was used to determine the intergroup differences. All tests were two‐sided. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 . Note: LPS: lipopolysaccharide; TNF‐α: tumor necrosis factor alpha; MCP‐1: monocyte chemoatractant protein‐1; IL‐10: interleukin‐10.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: In Vitro, Binding Assay, Pull Down Assay, Confocal Microscopy, Co-Immunoprecipitation Assay, Expressing, Standard Deviation

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: The aortic aneurysm, atherosclerosis, and inflammatory responses in ApoE −/− mice, sost −/− .ApoE −/− mice, ApoE −/− .Lrp8m and ApoE −/− . Lrp8m/Mac‐Lrp8 mice, with AngII infusion. (A) The diagram of experimental design. (B) Representative images of aortas in ApoE −/− mice, sost −/− .ApoE −/− mice, ApoE −/− .Lrp8m and ApoE −/− . Lrp8m/Mac‐Lrp8 mice with AngII infusion (scale bars, 4 mm). (C) Bar charts of the aortic aneurysm (AA) incidence. * P < 0.05 , ** P < 0. 01 for intergroup comparison by Fisher's exact test. (D) Representative images for en face Oil Red O staining of aortic arches (upper). Quantification of atherosclerotic plaque (lower). (E) Ex vivo measurement of the maximum diameters of aortic arches and suprarenal aortas. (F) The serum levels of inflammatory cytokine (TNF‐α), chemokine (MCP‐1), and anti‐inflammatory cytokine (IL‐10). (G) Representative immunofluorescence (IF) images for expression of iNOS (green) and CD206 (red) in paraffin sections of aortic roots (white dotted line, cell nucleus: blue). Scale bar: 200 µm (*lumen). (H) Quantification of the ratio of M2 anti‐inflammatory macrophage phenotype to M1 pro‐inflammatory macrophage phenotype in paraffin sections of aortic roots. (I) Representative Immunohistochemistry (IHC) images for phospho‐NF‐κB (p‐p65) in paraffin sections of aortic roots (black dotted line). Scale bar: 200 µm (*lumen). (J) Quantification of the ratio of phospho‐NF‐κB (p‐p65)‐positive cells to total cells in paraffin sections of aortic roots. Data were expressed as mean ± standard deviation. n = 12 per group. # P < 0.05 , ## P < 0.01 , ### P < 0.001 and #### P < 0.0001 for a comparison versus ApoE −/− + saline group by unpaired t‐test. * P < 0.05 , ** P < 0. 01 , *** P < 0.001 and **** P < 0.0001 for a comparison versus ApoE −/− + AngII by one‐way ANOVA with Tukey's post‐hoc test. ^ P < 0.05 , ^^ P < 0.01 , ^^^ P < 0.001 and ^^^^ P < 0.0001 for a comparison between ApoE −/− . Lrp8m/Mac‐Lrp8 + AngII group and ApoE −/− .Lrp8m + AngII group by unpaired t‐test. All tests were two‐sided. Note: AngII: Angiotensin II; TNF‐α: tumor necrosis factor alpha; MCP‐1: monocyte chemoattractant protein‐1; IL‐10: interleukin‐10; iNOS: inducible nitric oxide synthase (pro‐inflammatory macrophages biomarker); CD206: cluster of differentiation 206, known as mannose receptor C‐type 1 (anti‐inflammatory macrophages biomarker).

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Comparison, Staining, Ex Vivo, Immunofluorescence, Expressing, Immunohistochemistry, Standard Deviation, Saline, Biomarker Discovery

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: The aortic aneurysm, atherosclerosis, and inflammatory responses in sost −/− . ApoE −/− mice and sost −/− . ApoE −/− .Lrp8m mice, with and without rAAV8‐ mediated re‐expression of SOST or SOST loop2m . (A) The diagram of experimental design. (B) Representative images of aortas in sost −/− .ApoE −/− .Lrp8m mice and sost −/− .ApoE −/− mice, with AngII infusion (scale bars, 2 mm). (C) Bar charts of the aortic aneurysm (AA) incidence. ns P > 0.05 , * P < 0.05 for a comparison versus Fisher's exact test. (D) Representative en face Oil Red O staining images of aortic arches (upper) and quantification of atherosclerotic plaque (lower). (E) Ex vivo measurement of the maximum diameters of aortic arches and suprarenal aortas. (F) The serum levels of pro‐inflammatory cytokine (TNF‐α), pro‐inflammatory chemokine (MCP‐1) and anti‐inflammatory cytokine (IL‐10). (G) Representative immunofluorescence (IF) images for expression of iNOS (green) and CD206 (red) in paraffin sections of aortic roots (white dotted line, cell nucleus: blue). Scale bar: 200 µm (*lumen). (H) Quantification of the ratio of M2 anti‐inflammatory macrophage phenotype to M1 pro‐inflammatory macrophage phenotype in paraffin sections of aortic roots. (I) Representative Immunohistochemistry (IHC) images for phospho‐NF‐κB (p‐p65) in paraffin sections of aortic roots (black dotted line). Scale bar: 200 µm (*lumen). (J) Quantification of the ratio of phospho‐NF‐κB (p‐p65)‐positive cells to total cells in paraffin sections of aortic roots. Data were expressed as mean ± standard deviation. n=12 per group. ns P > 0.05, * P < 0.05 , ** P < 0. 01 , *** P < 0.001 , and **** P < 0.0001 for the comparison versus Veh controls by two‐way ANOVA with Tukey's post‐hoc test. All tests were two‐sided. Note: AngII: Angiotensin II; TNF‐α: tumor necrosis factor alpha; MCP‐1: monocyte chemoattractant protein‐1; IL‐10: interleukin‐10; iNOS: inducible nitric oxide synthase (pro‐inflammatory macrophages biomarker); CD206: cluster of differentiation 206, known as mannose receptor C‐type 1 (anti‐inflammatory macrophages biomarker).

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Expressing, Comparison, Staining, Ex Vivo, Immunofluorescence, Immunohistochemistry, Standard Deviation, Biomarker Discovery

Journal: Advanced Science

Article Title: Macrophagic Sclerostin Loop2‐ApoER2 Interaction Required by Sclerostin for Cardiovascular Protective Action

doi: 10.1002/advs.202518735

Figure Lengend Snippet: Schematic diagram showing our findings that sclerostin loop2‐ApoER2 interaction in macrophages was required by sclerostin to suppress NF‐κB nuclear translocation and phosphorylation, to promote macrophage conversion into anti‐inflammatory phenotypes in atherosclerotic aortas, as well as to prevent atherosclerosis and aortic aneurysm development in ApoE −/− mice.

Article Snippet: Study 2: Determination of whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro To determine whether the suppressive effects of sclerostin on inflammatory responses in macrophages were dependent on ApoER2 in vitro, PMA (100 ng/mL, 48 h)‐induced human differentiated macrophages (THP‐1 cells, Procell Life Science & Technology, CL‐0233) and mouse macrophages (RAW 264.7 cells, ATCC, TIB‐71) were utilized.

Techniques: Translocation Assay, Phospho-proteomics