caspase 8 polyclonal antibody  (Proteintech)

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 concurrently exist in human atherosclerotic lesions. (A–C) Necrotic core formation, lipid deposition and extracellular fibrosis are observed in lower extremity and carotid atherosclerotic lesions, but not in histologically normal artery by means of HE, Oil Red O, and Movat's staining. Scale bar: 1000 µm. (D) Immunohistochemical staining for CD68, a marker for macrophages, is conducted on histologically normal arteries as well as on lower extremity and carotid atherosclerotic lesions. Scale bar: 100 µm. (E–G) Compared with histologically normal artery (E), lower extremity (F) and carotid atherosclerotic lesions (G) show the pyroptotic, apoptotic and necroptotic characteristics of macrophages, including discontinuity of plasma membrane (red arrows), chromatin condensation, and electron‐light zones in transmission electron microscopy images. Scale bar: 2.5 µm. (H) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in human atherosclerotic lesions and peripheral normal artery. (I) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in human atherosclerotic lesions and peripheral normal artery. (J) The capacity of caspase‐3 in human atherosclerotic lesions and peripheral normal artery. (K and L) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in human atherosclerotic lesions and peripheral normal artery. (M) Representative Western blots and relative quantitative analysis of TLR4, MyD88 and phospho‐NF‐kB in human atherosclerotic lesions and peripheral normal artery. (N) Human atherosclerotic lesions release a significant amount of TNF‐1α, IL‐1β, IL‐18 and IL‐6, whereas the peripheral normal artery releases less. (O) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (yellow) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the co‐existence of pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Triple‐positive cells are shown by the arrows. Scale bar: 50 µm. (P–R) Double immunofluorescence staining for GSDMD (M)/caspase‐3 (N)/RIPK3 (O) (red), CD68 (green), and DAPI (blue) in human atherosclerosis and peripheral normal artery demonstrates the presence of pyroptotic, apoptotic and necroptotic markers in macrophages, as indicated by the colocalisation of caspase‐3/GSDMD/RIPK3 and CD68 (a macrophage marker). Double‐positive cells are shown by the arrows. Scale bar: 50 µm. 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: Staining, Immunohistochemical staining, Marker, Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Double 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: 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: Pyroptosis, apoptosis and necroptosis in macrophages concurrently exist in human atherosclerotic lesions. (A–C) Necrotic core formation, lipid deposition and extracellular fibrosis are observed in lower extremity and carotid atherosclerotic lesions, but not in histologically normal artery by means of HE, Oil Red O, and Movat's staining. Scale bar: 1000 µm. (D) Immunohistochemical staining for CD68, a marker for macrophages, is conducted on histologically normal arteries as well as on lower extremity and carotid atherosclerotic lesions. Scale bar: 100 µm. (E–G) Compared with histologically normal artery (E), lower extremity (F) and carotid atherosclerotic lesions (G) show the pyroptotic, apoptotic and necroptotic characteristics of macrophages, including discontinuity of plasma membrane (red arrows), chromatin condensation, and electron‐light zones in transmission electron microscopy images. Scale bar: 2.5 µm. (H) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in human atherosclerotic lesions and peripheral normal artery. (I) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in human atherosclerotic lesions and peripheral normal artery. (J) The capacity of caspase‐3 in human atherosclerotic lesions and peripheral normal artery. (K and L) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in human atherosclerotic lesions and peripheral normal artery. (M) Representative Western blots and relative quantitative analysis of TLR4, MyD88 and phospho‐NF‐kB in human atherosclerotic lesions and peripheral normal artery. (N) Human atherosclerotic lesions release a significant amount of TNF‐1α, IL‐1β, IL‐18 and IL‐6, whereas the peripheral normal artery releases less. (O) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (yellow) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the co‐existence of pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Triple‐positive cells are shown by the arrows. Scale bar: 50 µm. (P–R) Double immunofluorescence staining for GSDMD (M)/caspase‐3 (N)/RIPK3 (O) (red), CD68 (green), and DAPI (blue) in human atherosclerosis and peripheral normal artery demonstrates the presence of pyroptotic, apoptotic and necroptotic markers in macrophages, as indicated by the colocalisation of caspase‐3/GSDMD/RIPK3 and CD68 (a macrophage marker). Double‐positive cells are shown by the arrows. Scale bar: 50 µm. 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: Staining, Immunohistochemical staining, Marker, Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Double 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: 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: Nature Communications

Article Title: Prussian blue nanoparticles targeting multiple PANoptosome-mediated PANoptosis for myocardial ischemia-reperfusion injury therapy

doi: 10.1038/s41467-026-70012-2

Figure Lengend Snippet: A Schematic of ischemic zone (IZ), remote zone (RZ), and border zone (BZ) in human acute myocardial infarction. The heart element in the image was sourced from BioRender (Created in BioRender. xu, L. (2026) https://BioRender.com/9kka4p3 ). B UMAP visualization of cell distribution (left) and annotated cell types (right) in the control ( n = 4) versus IZ ( n = 11) groups. C Cardiomyocyte subpopulation quantification (left) and UMAP-based clustering (right) in the control and IZ groups. D , E UMAP projection ( D ) and box plots ( E ) showing PANoptosis activation across cardiac cell types. (The exact n in E = [left to right] 20, 84 cells; 17326, 3506 cells; 6231, 6969 cells; 9468, 12187 cells; 3386, 7142 cells; 3039, 2286 cells; 583, 254 cells; 531, 1138 cells). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right: 2.39E-265, 1.55E-300, 1.05E-35, 4.09E-05, 2.21E-06, 0.0002, 1.9E-11. F Violin plots comparing PANoptosis-related gene expression ( AIM2, ZBP1, RIPK1, Pyrin, NLRP12, NLRP3, MLKL, GSDMD, RIPK3, caspase 3, caspase 1 , and caspase 8 ) in total cells. ( n = 40706 cells [control], 33688 cells [IZ]). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right ( AIM2 to caspase 8 ): 0.2752, 0.0002, 0, 0.0854, 6.67E-09, 7.19E-82, 3.59E-37, 0.0709, 0.0269, 2.29E-280, 2.45E-30, 4.07E-09. G, H Cardiomyocyte-specific UMAP ( G ) and quantitative PANoptosis levels ( H ) of the control and IZ groups. (The exact n in H = [left to right] 6904 cells, 0; 5281 cells, 0; 1191, 3346 cells; 3192, 80 cells; 758, 80 cells). The minima, maxima, mean, median, bounds, whiskers, and percentile information were provided in the Source Data file. Exact P- values from left to right: 4.01E-05, 0.0021. I Proportional representation of cardiomyocyte subpopulations. J , K Heatmap depicting activation patterns of apoptosis/PANoptosis/pyroptosis/necroptosis ( J ) and PANoptosis-related genes ( K ) across cardiomyocyte subtypes. Significance: wilcox.test, a two-sided test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns : no significant difference ( P > 0.05). Source data are provided as a Source Data file.

Article Snippet: CD41 (24552-1-AP), Collagen III (22734-1-AP), AIM2 (20590-1-AP), caspase 1 (81482-1-RR), and caspase 8 (13423-1-AP) antibodies were bought from Proteintech, China.

Techniques: Control, Activation Assay, Gene Expression