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

Journal: Non-coding RNA Research

Article Title: MicroRNA-541-3p/Rac2 signaling bridges radiation-induced lung injury and repair

doi: 10.1016/j.ncrna.2025.01.010

Figure Lengend Snippet: Regulation of TNFRSF10B-RIPK1/RIPK3-MLKL signaling and necroptosis by microRNA-541-3p (A) Necroptosis in MLE-12 cells transfected with microRNA-541-3p. The left panel showed a flow cytometric analysis of MLE-12 cells transfected with microRNA-541-3p. The right panel showed the quantitative analysis of PE-positive cells. ∗ represented p < 0.05 when compared with the Nec- (intra-group). # represented p < 0.05 when compared with the control group. (B) Tnfrsf10b, Ripk1, Ripk3, and Mlkl genes were expressed in MLE-12 cells transfected with microRNA-541-3p. ∗ represented p < 0.05 when compared with the control group. (C) TNFRSF10B, RIPK1, RIPK3, and MLKL proteins were expressed in MLE-12 cells transfected with microRNA-541-3p. The left panel showed the band images after protein electrophoresis. The right panel showed the quantitative analysis of the relative gray value. ∗ represented p < 0.05 when compared with the control group. Three independent experiments were performed.

Article Snippet: We added 5 μL of Annexin V-FITC (AO2001-02P-H, Sungene) to the resuspended cells and incubated them in the dark for 10 min, then added 5 μL of PI and incubated them in the dark for 5 min. Flow cytometry was performed within 1 h. Methods to assess necroptosis: We added Nec-1 (Necrostain-1 [ ], a specific inhibitor of necroptosis, 20 μmol/L, Cas: 4311-88-0, TargetMol) to the culture medium and analyzed the effect of Nec-1 on the proportion of PE-positive cells.

Techniques: Transfection, Control, Protein Electrophoresis

Journal: Non-coding RNA Research

Article Title: MicroRNA-541-3p/Rac2 signaling bridges radiation-induced lung injury and repair

doi: 10.1016/j.ncrna.2025.01.010

Figure Lengend Snippet: The regulation of TNFRSF10B-RIPK1/RIPK3-MLKL signaling and necroptosis by Rac2 (A) Necroptosis in MLE-12 cells transfected with Rac2. The left panel showed a flow cytometric analysis of MLE-12 cells transfected with Rac2. The right panel showed the quantitative analysis of PE-positive cells. ∗ represented p < 0.05 when compared with the Nec- (intra-group). # represented p < 0.05 when compared with the control group. (B) Tnfrsf10b, Ripk1, Ripk3, and Mlkl genes were expressed in MLE-12 cells transfected with Rac2. ∗ represented p < 0.05 when compared with the control group. (C) TNFRSF10B, RIPK1, RIPK3, and MLKL proteins were expressed in MLE-12 cells transfected with Rac2. The left panel showed the band images after protein electrophoresis. The right panel showed the quantitative analysis of the relative gray value. ∗ represented p < 0.05 when compared with the control group. Three independent experiments were performed.

Article Snippet: We added 5 μL of Annexin V-FITC (AO2001-02P-H, Sungene) to the resuspended cells and incubated them in the dark for 10 min, then added 5 μL of PI and incubated them in the dark for 5 min. Flow cytometry was performed within 1 h. Methods to assess necroptosis: We added Nec-1 (Necrostain-1 [ ], a specific inhibitor of necroptosis, 20 μmol/L, Cas: 4311-88-0, TargetMol) to the culture medium and analyzed the effect of Nec-1 on the proportion of PE-positive cells.

Techniques: Transfection, Control, Protein Electrophoresis

Journal: Nature immunology

Article Title: Allergen protease-activated stress granule assembly and gasdermin D fragmentation control interleukin-33 secretion

doi: 10.1038/s41590-022-01255-6

Figure Lengend Snippet: a, Live-cell imaging of IL-33-GFP and NLS-GFP in A549 cells exposed to 5 μg of papain for the indicated time. Scale bars, 15 μm. b, GFP intensity statistics (ImageJ) of IL-33-GFP expression and NLS-GFP expression as in a. c, Immunoblot analysis of IL-33 and Gsdmd in MLE-12 cells after papain (1 μg, 5 μg, 10 μg and 50 μg) exposure for 30 min. Ctrl, no stimulation. d, ELISA analysis of IL-33 in WCL of MLE-12 cells stimulated with papain for 30 min. e, Immunoblot analysis of IL-33 and Gsdmd in MLE-12 cells after 5 μg well−1 papain exposure for the indicated time. f, ELISA analysis of IL-33 in WCL from MLE-12 cells with 5 μg well−1 papain exposure for the indicated time. Representative of four independent experiments. p.i., post infection. g, Flow cytometry analysis of MLE-12 cell survival with indicated stimulations. Ctrl, no stimulation; Pap, exposure to 10 μg of papain for 30 min; TS, TNF-α+SM-164 stimulation for 12 h; ns, not significant. Live cell, PI−annexinV−; apoptotic cell, PI−annexinV+; necroptotic cell, PI+annexinV+. h, Immunoblot analysis of MLE-12 cells stimulated with papain (5 μg) for 30 min followed by removal of papain for the indicated time. i, Immunoblot analysis of MLE-12 cells stimulated with papain (Pap, 100 μg well−1) for 30 min, TSZ (TNF-α+SM-164+Z-VAD-FMK) for 12 h and TS (TNF-α+SM-164) for 12 h. Red arrows indicate the functional p40 NT-Gsdmd. Nonspecific fragments marked with an asterisk were not discussed in our work. Error bar, mean ± s.e.m. b, n = 2, using a two-tailed, unpaired Student’s t-test; d, n = 4, f, n = 2, g, n = 3, with Dunnett’s multiple comparisons test. All data are representative of at least three independent experiments.

Article Snippet: The Necroptosis Inducer Kit with TSZ and the Apoptosis Inducer Kit (TNF-α + SM-164) (Beyotime Biotechnology, C1058S and C0006S) were used, as well as nuclear and cytoplasmic protein extraction kits (Beyotime Biotechnology, P0027; Merck Millipore, 539790).

Techniques: Live Cell Imaging, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Flow Cytometry, Functional Assay, Two Tailed Test