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: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Gal3-based duality in β 1 integrin dynamics. ( A ) Schematic representation of the molecular organization of Gal3 where the C-terminal carbohydrate recognition domain (CRD) and the N-terminal oligomerization domain are indicated. ( B ) Left: Representative region of interest (leading edge) of a 2D STORM image of an RPE-1 cell showing surface-bound Gal3 ( top ) and the corresponding clusters obtained after segmentation ( bottom ). Right : The occurrence of each type of Gal3 cluster is shown in function of their surface area (×10 3 nm 2 ). Means ± SEM, one-way ANOVA; ns = p > 0.05, **** p < 0.0001. Scale bar = 5 μm. ( C ) The percentage of Gal3 molecules is shown in function of the different Gal3 cluster populations. Means ± SEM, one-way ANOVA; ns = p > 0.05, * p < 0.05, ** p < 0.002, **** p < 0.0001. ( D ) 2D STORM image of Gal3 and anti-β 1 integrin antibodies on RPE-1 cells (4 °C co-binding). A zoom of the leading edge is shown to illustrate the extensive level of colocalization between Gal3 and β 1 integrin. Gal3 clusters with variable size and shape are detected (yellow arrowheads in the zoom). Scale bars = 5 μm. (E) Quantification of the probability of proximity (colocalization) between Gal3 and β 1 integrin for each class of Gal3 clusters, compared to random non-clustered Gal3. Means ± SEM, one-way ANOVA; ns = p > 0.05, **** p < 0.0001. ( F ) Top: Anti-β 1 integrin antibody uptake assay in RPE-1 cells. Internalized antibody is immunolabeled, imaged by confocal microscopy, and quantified. Bottom: Scheme of the protocol detailing the use of the Gal3 inhibitor I3 (10 μM) in acute versus prolonged incubation conditions. ( G ) Anti-β 1 integrin antibody uptake assay as in ( F ). Note the shift from both peripheral (green arrowheads) and perinuclear (red arrowheads) distribution of internalized β 1 integrin in control cells to exclusive perinuclear localization in the prolonged incubation condition. ( H ) Transferrin (Tf) internalization is very little affected by I3. In ( G ) and ( H ): Dashed lines indicate the contour of individual cells. Scale bars = 10 μm. Nuclei in blue (DAPI). Quantification of fluorescence intensities as means ± SEM, one-way ANOVA; ns = p > 0.05, ** p < 0.002, **** p < 0.0001.

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Binding Assay, Immunolabeling, Confocal Microscopy, Incubation, Control, Fluorescence

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: GSL-based duality in β 1 integrin dynamics. ( A ) Simplified schematic representation of the early steps of GSL synthesis. The reaction inhibited by Genz-123346 is indicated. ( B ) Analysis of cellular levels of the indicated GSL in function of incubation time with Genz-123346. Note that the most important drop occurs up to day 3. Means ± SEM, unpaired t -test; ns = p > 0.05, ** p < 0.002, **** p < 0.0001. ( C ) Scheme of experimental procedure detailing how GSL inhibition has been set up either in acute (3 days) or prolonged (5 days) incubation conditions, prior to cargo protein internalization for 10 min. ( D ) Anti-β 1 integrin antibody uptake assay as in ( C ). Note that β 1 integrin uptake is inhibited upon acute Genz-123346 treatment and increased upon prolonged treatment. In the latter condition, the intracellular accumulation of β 1 integrin is massively perinuclear (red arrowheads), compared to control cells where peripheral localizations are also observed (green arrowheads). Means ± SEM, unpaired t -test; **** p < 0.0001. ( E ) Transferrin (Tf) internalization (10 min) is only mildly affected in all conditions. Means ± SEM, unpaired t -test; ns = p > 0.05, ** p < 0.002. ( F ) Internalization of exogenous Gal3 (10 min). Similar to β 1 integrin, Gal3 endocytosis is significantly inhibited upon acute Genz-123346 treatment, and increased with perinuclear accumulation upon prolonged treatment (red arrowheads). Means ± SEM, unpaired t -test; **** p < 0.0001. In ( D – F ): Yellow dashed lines indicate contours of cells; scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Incubation, Inhibition, Control

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Characterization of sites of perinuclear β 1 integrin accumulation. ( A , B ) Anti-β 1 integrin or ( C ) Gal3 uptake assay (10 min) under acute or prolonged I3 ( A ) or Genz-123346 ( B , C ) treatment followed by immunolabeling for EEA1. The colocalization of β 1 integrin ( A , B ) or Gal3 ( C ) with EEA1 as well as the fluorescent intensity of EEA1 signal were quantified ( right ). Note the increased colocalization of internalized β 1 integrin ( A , B ) or Gal3 ( C ) with EEA1 and increased EEA1 signal intensity, notably in the prolonged treatment conditions. Means ± SEM, one-way ANOVA ( A , B ), or unpaired t -test ( C ); **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Immunolabeling

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Exogenous Gal3 and dextran 70K uptake upon prolonged GSL depletion. After prolonged (5 days) treatment with Genz-123346, RPE-1 cells were continuously co-incubated (10 min) with exogenous Gal3 and dextran 70K. Note the increased perinuclear accumulation of Gal3 and its increased overlap with dextran 70K under these conditions. Means ± SEM, unpaired t -test; **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Incubation

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Role of clathrin in endocytic uptake under prolonged treatment conditions. ( A – C ) Uptake assays (10 min) of anti-β 1 integrin antibodies ( A , B ) or Gal3 ( C ) upon prolonged I3 ( A ) or Genz-123346 ( B , C ) treatment. When indicated (siCHC), clathrin heavy chain was depleted ( right images). The perinuclear accumulation of β 1 integrin ( A , B ) or that of Gal3 ( C ) as observed in the prolonged treatment conditions (red or white arrowheads) is strongly inhibited upon clathrin depletion. Means ± SEM, one-way ANOVA; **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques:

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Continuum model between lattices and GL-Lect driven endocytosis. ( A ) Unperturbed condition. A glycoprotein cargo, here α 5 β 1 integrin, is either recruited into galectin lattices ( left , underlined in red) or internalized by GL-Lect driven endocytosis ( right , underlined in blue). ( B ) Acute treatment conditions. Since tubular endocytic pits for GL-Lect driven endocytosis are built de novo, acute interference with Gal3 activity or GSL expression prevents their formation. In contrast, preassembled galectin lattices resist under these conditions. ( C ) Prolonged treatment conditions. Even galectin lattices are disassembled. With GL-Lect driven endocytosis being inhibited, α 5 β 1 integrin is now internalized by alternative endocytic pathways, i.e., clathrin-mediated endocytosis and macropinocytosis.

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Activity Assay, Expressing