1000 p ikbα af1870 biyuntian 1  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis is activated in MFCs in fat graft tissue. (A) TEM of the ultrastructure of MFCs located in fat tissue after grafting. N, nucleus; LD, lipid droplet; PS, pseudopodia; M, mitochondria; RER, rough endoplasmic reticulum; and Lys, lysosome. Red arrowheads indicated the damaged plasma membrane. (B,C) Representative pMLKL staining (B) and quantification of the pMLKL-positive area (C) in fat tissue before and after grafting. (D) Representative immunofluorescence staining of pMLKL (red) and F4/80 (green) in fat tissue after grafting. Scale bars = 50 μm. Data are presented as the mean ± SD. ## P < 0.01 compared with the pre-grafting group; ** P < 0.01.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Clinical Proteomics, Membrane, Staining, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis is activated in MFCs in the in vitro CLS cell culture model. (A,B) Necrosis of macrophages determined by Hoechst 33342 and PI staining in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. (A) Hoechst 33342 + PI + macrophages in the pictures indicate necrotic macrophages. (B) Quantification of macrophage necrosis in both groups at 24 and 96 h. (C,D) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. (E) Immunofluorescence staining of pMLKL (red) and F4/80 (green) in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. Scale bars = 50 μm. A, adipocyte; M, macrophage. Data are presented as the mean ± SD. ## P < 0.01 compared with the group of RAW 264.7 macrophages cultured alone at 24 h.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: In Vitro, Cell Culture, Staining, Western Blot, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis inhibitors suppressed necroptosis of MFCs in the in vitro CLS cell culture model. (A,B) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in four groups, including RAW 264.7 macrophages cultured alone, and those in CLS cell culture incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872) at 96 h. (C,D) Necrosis of macrophages determined by Hoechst 33342 and PI staining in four groups of RAW 264.7 macrophages cultured alone, and those in CLS cell culture incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872) at 96 h. Scale bars = 50 μm. Data are presented as the mean ± SD. ## P < 0.01 compared with the CLS cell culture model group incubated without necroptosis inhibitors.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: In Vitro, Cell Culture, Western Blot, Incubation, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis of MFCs induces collagen expression in fibroblasts via a paracrine mechanism. (A,B) MILLIPLEX MAP assays were used to detect the expression levels of cytokines/chemokines in conditioned media collected from six groups, including adipocytes (live or apoptotic), RAW 264.7 macrophages, and CLS cultured cells incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872). (C) Fibroblasts were treated with conditioned media collected from the six groups. (D) Immunofluorescence staining of collagen I and collagen VI in fibroblasts to investigate the paracrine effect of the culture media from various groups on fibroblasts. (E) Quantification of the collagen I- and collagen VI-positive areas per fibroblast. Scale bars = 50 μm. Data are presented as the mean ± SD. # P < 0.05, ## P < 0.01 compared with the CLS cell culture model group incubated without necroptosis inhibitors.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Expressing, Cell Culture, Incubation, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Blockade of necroptosis alleviates fat graft fibrosis. (A) Illustration of the animal model. Fat grafting model mice were administered Nec-1 or vehicle. (B) Macroscopic views of harvested fat tissue. (C) The retention volume of the fat grafts over time. (D–F) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in vivo . (G) The gene expression level of the cytokines TNF-α and IL-6 and the chemokines MCP-1 and MIP-2 in fat grafts. (H) Representative Masson’s trichrome staining of fat tissue at week 16 after grafting in the different groups. (I) Representative Sirius red staining of fat tissue at week 16 after grafting in the different groups. (J–L) Quantification of the areas positive for Masson’s trichrome (J) , collagen I (K) , and collagen VI (L) staining in fat tissue at week 16 after grafting in the different groups. Scale bars = 50 μm. Data are presented as the mean ± SD. # P < 0.05, ## P < 0.01 compared with the control group. (M) TEM analyses of the ultrastructure of MFCs in fat tissue at week 16 after grafting in the different groups. N, nucleus; LD, lipid droplet; PS, pseudopodia; M, mitochondria; RER, rough endoplasmic reticulum; Go, golgiosome; Lys, lysosome; and Red arrowheads indicated the damaged plasma membrane.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Animal Model, Western Blot, In Vivo, Gene Expression, Staining, Control, Clinical Proteomics, Membrane

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Potential role of MFC necroptosis in fat graft fibrosis. After fat grafting, lipids released from apoptotic adipocytes induce formation of MFCs and an increased level of cellular lipids mediates necroptosis of MFCs, which upregulates the expression of proinflammatory cytokines/chemokines, leading to collagen synthesis by fibroblasts. Consequently, overproduction of collagens leads to fat tissue fibrosis after grafting.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Expressing