Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Identification of DEPs and DEGs by proteomic and transcriptome analysis of the aortas of ApoE −/− mice versus HFD‐fed ApoE −/− mice. (A) Volcano plot analysis represents the up‐ or downregulated DEPs abundance changes in the aortas from ApoE −/− mice versus HFD‐fed ApoE −/− mice. Blue indicates upregulated genes, green indicates downregulated genes, and grey indicates genes with unchanged genes. (B) The top six upregulated DEPs consisting of lgals3/galectin‐3, nucb2, ighm, vcam1, serpina1e and serpina3n between ApoE −/− mice and HFD‐fed ApoE −/− mice. (C) Numbers of DEGs are enriched in the identified pathway. Apoptosis is identified as the critical cell death forms. The x ‐axis represents the number of enriched genes, and the y ‐axis represents the name of the enriched KEGG pathway. (D) Compared to ApoE −/− mice, the gene set upregulated in HFD‐fed ApoE −/− mice is enriched in the signal pathways including foal adhesion, leukocyte transendothelial migration, Toll‐like receptor signalling pathway, natural killer cell mediated cytotoxicity, and apoptosis. (E) The Volcano plot illustrates that gene transcripts with a log2 fold change greater than 1 and a significant p ‐value less than.05 are differently expressed between normal and atherosclerosis. Red represents upregulation, blue represents downregulation, and grey represents no change. (F) KEGG enrichment of DEGs indicates that apoptosis emerged as the fifth pathway of significant alteration. KEGG pathway analysis is performed with bar plot. Colours correspond to the p ‐value, with red indicates more significant enrichment. (G) Venn diagram is performed to screen out the overlapping DEPs and DEGs. (H) The coordinated DEPs and DEGs, consisting of Galectin/Lgals3, Vcam1 and Ctss, are differentially expressed between ApoE −/− control mice and HFD‐fed ApoE −/− mice.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Migration, Control

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Molecular classification of human carotid atherosclerotic database based on PANoptosis‐related genes. (A) The CDF curves of consensus matrix for k = 2–5 are illustrated using distinct colours. K value represented the number of clusters. (B) The line graph of CDF area under curve is depicted at k = 2–5. The curve area with minimal variation is between k = 2 and k = 1, thus the clustering effect is relatively stable when k = 2. (C) Given that consensus matrix with k = 2 is an optimal choice, the entire cohort is separated into two clusters. (D) Item consensus plot when k = 2 indicates that the cluster pattern shared the acceptable level of purity in both clusters. (E) The distribution of immune, stromal and overall ESTIMATE scores is inferred by ESTIMATE algorithm between two clusters in the GSE111782 cohort, and three kinds of scores are substantially greater in Cluster 1 than Cluster 2. (F) The infiltration abundance of macrophage subsets is evaluated by CIBERSORT algorithm for two clusters, and Cluster 1 had a greater proportion of macrophage M1 and M2 than Cluster 2. (G) Landscape of PANoptosis‐related gene expression is depicted in Cluster 1 and Cluster 2. (H) KEGG enrichment of all DEGs shows the top 10 signalling pathways. The colour and size of each bubble indicate p ‐value and gene count, respectively. (I) Landscape of three overlapping DEGs and DEPs, including Lgals3/galectin‐3, Vcam1 and Ctss is depicted in Cluster 1 and Cluster 2. Three key DEGs and DEPs are predicted to be activated in Cluster 1 compared to Cluster 2. (J) A PPI network of the three DEGs and DEPs, and 11 PANoptosis‐related genes is created according to the STRING database.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Gene Expression

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Galectin‐3 expression is abundant in human and mouse atherosclerotic lesions. (A) Ten main cell types are visualised in atherosclerotic core (AC) and proximal adjacent (PA) tissues by tSNE (t‐distributed stochastic neighbour embedding). (B) The macrophage population significantly increased in AC relative to PA. (C) Biaxial scatter plots show the expression pattern of galectin‐3 in total cell types between AC and PA. The colour scale represents expression levels in biaxial scatter plots (grey: low; pink: high). (D) Galectin‐3‐positive macrophages expanded in AC in comparison with PA. (E) Five macrophage subtypes are visualised in AC and PA tissues by tSNE. (F) My.0 and My.1 account for 34.1% and 47.6% of macrophages in AC, respectively. My.2 significantly increased in AC relative to PA. (G) Biaxial scatter plots exhibit the expression pattern of galectin‐3 in macrophage subtypes between AC and PA. (H) Galectin‐3‐positive My.0 and My.1 account for 35.8% and 47.5% of galectin‐3‐positive macrophages in AC, respectively. Galectin‐3‐positive My.2 expands in AC in comparison with PA. (I) Representative Western blots and relative quantitative analysis of galectin‐3 in human atherosclerotic lesions and peripheral normal artery. (J) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the colocalisation of galectin‐3 and NLRP3 in CD68‐positive macrophages. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of galectin‐3 in the aortas of ApoE −/− mice fed with an HFD or normal diet. (L) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals that galectin‐3 and NLRP3 are colocalised in CD68‐positive macrophages. Scale bar: 50 µm. (M) Cell lysates from ox‐LDL‐treated macrophages are immuno‐precipitated with anti‐galectin‐3 or anti‐NLRP3 antibodies, and blotted with anti‐NLRP3 or anti‐galectin‐3 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Expressing, Comparison, Western Blot, Immunofluorescence, Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Gene Oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) in macrophage between atherosclerotic core (AC) and proximal adjacent (PA) tissues. (A) Volcano plot of DEGs in macrophages is conducted between PA and AC. Lgals3/galectin‐3 are identified as a critical DEG expressed in macrophages. (B) Enriched GO terms are depicted with DEGs in macrophages, including necroptotic signalling pathway, pyroptotic inflammatory response, regulation of apoptotic signalling pathway, MyD88‐dependent Toll‐like receptor 4 signalling pathway, and canonical NF‐kappyB signal transduction, and so forth. (C) KEGG analysis is conducted using DEGs in macrophages, including fluid shear stress and atherosclerosis, leukocyte transendothelial migration, pyroptosis, lipid and atherosclerosis, necroptosis, Toll‐like receptor signalling pathway, NF‐kB signalling, and apoptosis. The x ‐axis corresponds to the number of enriched DEGs, and the y ‐axis corresponds to the enriched pathway. Colours indicate the p ‐values, with red more significant enrichment. (D–F) Gene set of pyroptosis, apoptosis, necroptosis, NF‐kB signalling pathway, Toll‐like receptor signalling pathway, leukocyte transendothelial migration, lipid and atherosclerosis, and fluid shear stress and atherosclerosis are significantly upregulated in macrophages during atherosclerosis.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Transduction, Shear, Migration

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Silencing galectin‐3 downregulated TLR4/MyD88/NF‐kB expression and attenuated ox‐LDL induced pyroptotic, apoptotic, and necroptotic cell death in macrophages. (A) Electron microscopy ultrastructural analysis of control and ox‐LDL‐induced macrophages. Control macrophages have a normal‐looking cellular structure, whereas ox‐LDL‐induced macrophages show loss of cell plasma integrity, chromatin condensation or fragmentation, and electron‐light zone. Scale bar: 2.5 µm. (B) Confocal microscopy with double immunofluorescence staining for caspase‐3 (red) and RIPK3 (green) in macrophages show the colocalisation of apoptotic and necroptotic components. Confocal microscopy analysis of double immunofluorescence labelling is indicative of overlapping expression of caspase‐3 (red) and GSDMD (green) in macrophages. Scale bar: 25 µm. (C) Representative Western blots and relative quantitative analysis of galectin‐3 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (D and E) Flow cytometry (E) and quantification analysis (F) with annexin V/PI double staining show that ox‐LDL increased the percentage of apoptotic cells in macrophages, which is alleviated by silencing galectin‐3. (F–H) Flow cytometry (F) and quantification analysis (G) with PI/Hoechst staining (H) show that ox‐LDL enhanced PI uptake in macrophages, which is markedly blocked by silencing galectin‐3. Scale bar: 50 µm. (I) Silencing galectin‐3 abrogated LDH release in macrophages ignited by ox‐LDL. (J) Ox‐LDL induced the accumulation of intracellular lipid droplets in macrophages, which are potently reversed by silencing galectin‐3. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (L) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (M and N) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (O) Ox‐LDL treatment promotes the release of proinflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) from macrophages, which is markedly rescued by silencing galectin‐3. (P and Q) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐kB and phospho‐NF‐kB in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (R) Cell lysates from ox‐LDL‐treated macrophages are immunoprecipitated with anti‐TLR4 or anti‐MyD88 antibodies, and blotted with anti‐TLR4 or anti‐MyD88 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Expressing, Electron Microscopy, Control, Clinical Proteomics, Confocal Microscopy, Double Immunofluorescence Staining, Immunofluorescence, Western Blot, Flow Cytometry, Double Staining, Staining, Immunoprecipitation, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: NLRP3 agonist nigericin counteracted the inhibitory effect of silencing galectin‐3 on pyroptosis, apoptosis and necroptosis in macrophages. (A) Confocal microscopy with double immunofluorescence staining for galectin‐3 (red) and NLRP3 (green) in macrophages reveals the colocalisation of galectin‐3 with NLRP3. Scale bar: 25 µm. (B and C) Flow cytometry (B) and quantification analysis (C) with annexin V/PI double staining show that silencing galectin‐3 decreases the percentage of apoptotic cells in ox‐LDL‐induced macrophages, and nigericin robustly blunts the inhibitory effect of siGalectin‐3. (D–F) Flow cytometry (D) and quantification analysis (E) with PI/Hoechst staining (F) show that silencing galectin‐3 diminishes the percentage of PI‐positive cells in ox‐LDL‐induced macrophages, and nigericin mostly abolishes the protective effect of siGalectin‐3. Scale bar: 50 µm. (G) Silencing galectin‐3 suppresses the LDH release in ox‐LDL‐induced macrophages, which is largely abrogated by nigericin. (H) Silencing galectin‐3 lessens the intracellular lipid droplet in ox‐LDL‐induced macrophages, while nigericin exerts the opposite effect. Scale bar: 50 µm. (I) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (J) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (K) The activity of caspase‐3 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (L and M) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (N) Silencing galectin‐3 inhibits the release of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in ox‐LDL‐induced macrophages, and nigericin effectively blocks the role of siGalectin‐3. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Confocal Microscopy, Double Immunofluorescence Staining, Flow Cytometry, Double Staining, Staining, Western Blot, Activity Assay, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages coordinately occurred in ApoE −/− mice fed an HFD, which are alleviated by galectin‐3 deficiency, and conversely are aggravated by NLRP3 agonist nigericin. (A) Pyroptosis, apoptosis and necroptosis of macrophages are identified in the aortas of ApoE −/− mice fed an HFD, as evidenced by plasma membrane pore (red arrows), chromatin condensation (red arrows), and electron‐light zone (red arrows) by transmission electron microscopy. Scale bar: 2.5 µm. (B) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (pink) and DAPI (blue) in the aortas of ApoE −/− mice fed HFD or normal diet reveals the potential crosstalk among pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Three‐positive cells are shown by the arrows. Scale bar: 50 µm. (C–E) Dual immunofluorescence staining for caspase‐3 (C)/GSDMD (D)/RIPK3 (E) (red), F4/80 (green), and DAPI (blue) in the aortas of ApoE −/− mice fed an HFD or normal diet demonstrate that GSDMD/caspase‐3/RIPK3 immunoreactivity colocalises with macrophage marker CD68. Scale bar: 50 µm. (F) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in the aortas of ApoE −/− mice fed with a normal diet or HFD, NLRP3 agonist nigericin‐treated ApoE −/− mice fed with an HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (G) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase 8 and cleaved caspase 8 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with HFD. (H) The activity of caspase‐3 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (I and J) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (K and L) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐κB and phospho‐NF‐κB in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Marker, Western Blot, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Galectin‐3 genetic deficiency or knockdown reduced and, conversely, NLRP3 agonist nigericin augmented atherosclerotic lesions in HFD‐fed ApoE −/− mice. (A) The knockout efficacy of galectin‐3 in the aorta is verified by Western blotting and RT‐qPCR. (B) Schematic diagram of animal study design. Galectin‐3 −/− /ApoE −/− mice and ApoE −/− mice are fed an HFD for 16 weeks, and ApoE −/− mice are intraperitoneally administered with NLRP3 agonist nigericin. (C) Representative images of en face Oil Red O staining in the entire aortas are obtained from ApoE −/− control mice, HFD‐fed ApoE −/− mice, HFD‐fed ApoE −/− mice treated with nigericin, and HFD‐fed Galectin‐3 −/− /ApoE −/− mice. Scale bar: 50 mm. (D) En face lesion area is quantified as a percentage of the total area of the aorta. Compared with those in HFD‐fed ApoE −/− mice, en face lesion areas are significantly smaller in HFD‐fed Galectin‐3 −/− /ApoE −/− mice and, conversely, are markedly bigger in HFD‐fed ApoE −/− mice treated with nigericin. (E) Representative sections of HE, Oil Red O and Movat's staining in the aortic sinuses are acquired from four different groups of mice. Scale bar: 1 mm. (F) Aortic sinus plaque lesion area, lipid lesion area and mucin area are represented as total area in µm 2 . Plaque area, lipid lesion area (red) and mucin area (blue‐green) are much bigger in HFD‐fed ApoE −/− mice treated with nigericin and, conversely, are relatively smaller in HFD‐fed Galectin‐3 −/− /ApoE −/− mice in comparison with HFD‐fed ApoE −/− mice. (G) The levels of inflammatory cytokines in the aortas are measured from four different groups of mice. (H) Schematic illustration of experimental protocol in ApoE −/− mice receiving the injection of AAV‐F4/80 shGalectin‐3/empty vector at the age of 4 weeks. After 2 weeks of a normal diet for rest, these mice are treated with HFD for 16 weeks. (I) The knockdown efficacy of shGalectin‐3 in the aorta is confirmed through Western blotting and RT‐qPCR. (J) Representative images of en face Oil Red O staining in the entire aortas are obtained from HFD‐fed ApoE −/− mice, empty vector‐treated HFD‐fed ApoE −/− mice and shGalectin‐3‐treated HFD‐fed ApoE −/− mice. Scale bar: 50 mm. (K) En face lesion area, quantified as a percentage of total area of the aorta, is significantly smaller in shGalectin‐3‐treated HFD‐fed ApoE −/− mice than in empty vector‐treated HFD‐fed ApoE −/− mice. (L) Representative sections of HE, Oil Red O and Movat's staining in the aortic sinuses are acquired from three different groups of mice. Scale bar: 1 mm. (M) Aortic sinus plaque lesion area, lipid lesion area (red) and mucin area (blue‐green), represented as total area in µm 2 , are much bigger in shGalctin‐3‐treated HFD‐fed ApoE −/− mice than in empty vector‐treated HFD‐fed ApoE −/− mice. (N) The levels of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in the aortas are measured from three different groups of mice. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Membranes were then incubated overnight on a shaker with primary mouse or rabbit antibodies against galectin‐3 (60207‐1‐Ig, Proteintech, China), GSDMD (AF4012, Affinity Biosciences, China), NLRP3 (DF7438, Affinity Biosciences, China), caspase‐3 (66470‐2‐lg, Proteintech, China), caspase‐8 (66093‐1‐Ig, Proteintech, China), RIPK3 (A5431, ABclonal, China), MLKL (A26436, ABclonal, China), Phospho‐MLKL (AP0949, ABclonal, China), TLR4 (GB11519, Servicebio, China), MyD88 (GB12269, Servicebio, China), NF‐κB (10745‐1‐AP, Proteintech, China), Phospho‐NF‐κB ( GB113882 , Servicebio, China) and GAPDH (60004‐1‐Ig, Proteintech, China).

Techniques: Knockdown, Knock-Out, Western Blot, Quantitative RT-PCR, Staining, Control, Comparison, Injection, Plasmid Preparation

Journal: Cell Proliferation

Article Title: Glycomics reveal that ST6GAL1‐mediated sialylation regulates uterine lumen closure during implantation

doi: 10.1111/cpr.13169

Figure Lengend Snippet: Expression patterns of the sialylated glycans as well as the mRNA and protein expression of ST6GAL1 and ST6GAL2 in pig endometrium during implantation. (A) The 19 possible α2,3/6‐sialylated N‐glycans identified by MALDI‐TOF MS. Fucose ; N‐acetylglucosamine ; N‐acetyl neuraminic acid ; N‐glycolyl neuraminic acid ; mannose ; Galactose . (B) Representative images of lectin fluorescence assays taken from pig endometrium on gestational days 12, 15 and 18 (n = 3 gilts/gestational day). MAL‐II was used to detect α2,3‐linked sialic acid residues. SNA was used to detect α2,6‐linked sialic acid residues. (C) Expression levels of ST6GAL1 and ST6GAL2 in pig endometrium on gestational days 12, 15 and 18 measured by qRT‐PCR (n = 3 gilts/gestational day). The error bars represent the standard error. Mean is denoted by a red line. Nonparametric Mann‐Whitney one tail test was used for statistical analysis. (D) Distributions of ST6GAL1 and ST6GAL2 in pig endometrium on gestational days 12, 15 and 18 (n = 3 gilts/gestational day). The positive signal is in green, while the nucleus is in blue. NC, negative control. LE, endometrial luminal epithelium. GD, gestational day. Scale bar = 50 µm

Article Snippet: The primary antibodies used were ST6GAL1 (1:50, ab77676, abcam), ST6GAL2 (1:50, AF7747; R&D Systems), E‐cadherin (1:50, ab40772; abcam), β‐catenin (1:50, ab6302; abcam), Vimentin (1:50; sc‐73258), Rac1 (1:30, 66122–1‐Ig; Proteintech) and RhoA (1:50, 10749–1‐Ig; Proteintech).

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, MANN-WHITNEY, Negative Control

Journal: Gut Pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: In silico predicted conserved murine and human targets of miR-125a-5p and miR-615-3p. A The Venn diagram presents interfaces of overlapping targets of miR-125a-5p and miR-615-3p within the mucin-type O -glycosylation pathway of which the glycosyltransferases St3gal1 and B4galt1 are conserved in human and mouse, respectively. B Excerpt of the KEGG signalling pathway enrichment analysis of mucin-type O -glycosylation in mouse and human . Marked by the red asterisks are the specific steps in the glycosylation that might be modified by miR-125a-5p and miR-615-3p interaction. C Two binding sites of miR-125a-5p were identified in the 3’UTR of the murine St3gal1 using RNAhybrid

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: In Silico, Glycoproteomics, Modification, Binding Assay

Journal: Gut Pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Specific interaction between miR-125a-5p and St3gal1 was verified by RNAi and dual luciferase reporter assay. A Transfection efficiency of the murine intestinal cell line CMT 93 with miR-125a-5p mimics as well as expression of potential targets was evaluated by means of RT-qPCR. Most pronounced and significant decrease of the target St3gal1 was detected after miR-125a-5p transfection. B4galt1 showed significantly reduced levels. St3gal2 showed no significant difference. miR-615-3p showed upregulated but not significant expression. Non-target miRNA was used as a control, fold changes were calculated relatively to the non-target control and normalised with HPRT and SDHA or SNORD44 and SNORD 47, respectively. Charts indicate means ± standard deviations (SD) of three biological samples with triple measurements. B Relative luciferase activity was determined in comparison to control non-target miRNA mimic. miR-125a-5p caused clear and significant activity of the reporter gene fused to the target sites compared with non-target transfected controls. Charts indicate means ± SD of three biological replicates with technical triplicates. Statistical significance is presented by asterisks compared to negative controls at each time point. *P ≤ 0.05, **P ≤ 0.01, unpaired t-test

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR, Control, Activity Assay, Comparison

Journal: Gut Pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Relative gene expression of relevant miRNAs and their target mRNAs in secondary abiotic IL10 −/− mice colonic tissue sections six days post C. jejuni infection. 19 C. jejuni infected and 20 naïve control mice were included in the study. Each data point in the diagram represents an animal that showed confirmed expression in the respective analysis. A Upon C. jejuni infection, expression of miR-125a-5p was significantly decreased, whereas transcriptional levels of St3gal1 and B4galt1 were significantly enhanced. B miR-615-3p levels were significantly increased, while St3gal2 was significantly downregulated. Expressions were relatively calculated to naïve controls and normalised with SNORD44 and SNORD47 or with HPRT and SDHA, respectively. For each individual, the mean was calculated based on triplicate measurements. Charts show mean ± SD of all individuals. Statistical significance is presented by asterisks compared to naïve controls. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired t-test

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Gene Expression, Infection, Control, Expressing

Journal: Gut Pathogens

Article Title: miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis

doi: 10.1186/s13099-023-00577-6

Figure Lengend Snippet: Increased colonic ST3GAL1 protein levels in secondary abiotic IL10 −/− mice six days post C. jejuni infection compared to naïve controls. A Increased ST3GAL1 protein levels were detected by western blots in pooled protein samples of infected mice. GAPDH is presented as the respective loading reference. B ST3GAL1 detection of eight C. jejuni infected and eight naïve mice colon samples were quantified by means of western blotting followed by densitometric analysis relative to the respective GAPDH signals as controls. Protein levels of ST3GAL1 in the infected group were significantly and 1.7-fold increased, compared to naïve controls. Charts show normalised mean ± SD in each group. Statistical significance is presented by asterisks. **P ≤ 0.01, unpaired t-test. C Localisation and enhanced ST3GAL1 expression in the colon of C. jejuni infected mice determined in representative immunofluorescent staining compared to naïve controls. ST3GAL1 was detected by immunofluorescent staining and shown in red using DyLight 594 whereas the nuclei were stained blue using DAPI. Enhanced red signal intensity and pronounced cytosolic distribution was detected in infected controls. The top row shows the overview, with the area outlined in green in the bottom row shown enlarged. Scale bars indicate 100 µm and 50 µm. Exposure time was identical for all colon sections and presented images are representative for three biological replicates tested

Article Snippet: Then, the sections were incubated with a 1:50 dilution of the Rabbit anti-ST3GAL1 primary antibody (Novus Biologicals, NBP1-62540), in 1% (v/v) BSA in PBST overnight at 4 °C.

Techniques: Infection, Western Blot, Expressing, Staining