Journal: International Journal of Molecular Medicine

Article Title: Hypothermic machine perfusion protects DCD graft liver from ischemia-reperfusion injury by enhancing macrophage efferocytosis via KLF2-NLRP3 signaling

doi: 10.3892/ijmm.2026.5756

Figure Lengend Snippet: Upregulation of the endothelial cell protective molecule KLF2 promotes macrophage efferocytosis. (A) In vivo assessment and (B) quantification of efferocytosis (proportion of CD68-positive cells engulfing TUNEL-positive apoptotic cells relative to the total CD68-positive cell population). Arrows represent macrophage efferocytosis events. (C) Schematic diagram of the co-culture protocol for HUVECs with varying KLF2 expression levels and macrophages. (D) Macrophages (CMTPX + ) were co-cultured with apoptotic Jurkat cells (CMFDA + ) for 45 min, and efferocytosis was quantitatively assessed in vitro by fluorescence co-localization analysis. (E) Statistical analysis of fluorescence signals from HUVECS of ov-control and ov-KLF2 groups was performed using Image-Pro Plus 6.0 software (n=3). (F) Statistical analysis of fluorescence signals from HUVECS of sh-control and sh-KLF2 groups was performed using Image-Pro Plus 6.0 software (n=3). (G) Representative staining of TIMD4. (H) Immunofluorescence of TIMD4 from HUVECS of ov-control and ov-KLF2 groups was quantified using Image-pro Plus 6.0. (I) Immunofluorescence of TIMD4 from HUVECS of ov-control and ov-KLF2 groups was quantified using Image-pro Plus 6.0 (n=3). * P<0.05, ** P<0.01, *** P<0.001. HMP, hypothermic machine perfusion; KLF2, Kruppel-like Factor 2; AC, apoptotic Jurkat cell; HUVEC, human umbilical vein endothelial cells; CMTPX, CellTracker™ Red CMTPX dye; CMFDA, 5-Chloromethylfluorescein Diacetate; CS, cold storage; Rep, reperfusion; TIMD4, T-cell immunoglobulin and mucin domain-containing protein 4; ov, overexpression; sh, short hairpin.

Article Snippet: ACs were labeled with 5-chloromethylfluorescein diacetate (CMFDA) (cat. no. 40721ES) and macrophages were labeled with CellTrackerTM Red CMTPX dye (CMTPX) (cat. no. 40717ES; both Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacturer's instructions.

Techniques: In Vivo, TUNEL Assay, Co-Culture Assay, Expressing, Cell Culture, In Vitro, Fluorescence, Control, Software, Staining, Immunofluorescence, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: Hypothermic machine perfusion protects DCD graft liver from ischemia-reperfusion injury by enhancing macrophage efferocytosis via KLF2-NLRP3 signaling

doi: 10.3892/ijmm.2026.5756

Figure Lengend Snippet: Pyroptosis of ECs inhibits the occurrence of efferocytosis in macrophages. (A) Experimental protocol of nigericin treatment. (B) Morphology and structure of HUVECs treated with cell pyroptosis agonist nigericin were investigated by bright-field imaging (n=3). (C) Schematic diagram of the procedure for adding nigericin to the HUVEC-macrophage co-culture system. (D) Representative staining images showing efferocytosis in macrophages underwent non-contact co-culture with HUVECs treated with nigericin. (E) Quantitative analysis of efferocytosis in macrophages. (n=3). * P<0.05, ** P<0.01, *** P<0.001. AC, apoptotic Jurkat cell; HUVEC, human umbilical vein endothelial cell; CMTPX, CellTracker™ Red CMTPX dye; CMFDA, 5-Chloromethylfluorescein Diacetate.

Article Snippet: ACs were labeled with 5-chloromethylfluorescein diacetate (CMFDA) (cat. no. 40721ES) and macrophages were labeled with CellTrackerTM Red CMTPX dye (CMTPX) (cat. no. 40717ES; both Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacturer's instructions.

Techniques: Imaging, Co-Culture Assay, Staining

Journal: bioRxiv

Article Title: Efficient Inhibition of O-glycan biosynthesis using the hexosamine analog Ac 5 GalNTGc

doi: 10.1101/2020.12.13.422539

Figure Lengend Snippet: A. Mouse bone marrow cells (mBMC) and neutrophils (mPMN) were isolated from 10-12 week C57BL/6 mice, and cultured with 50μM HexNAc analogs or controls for 40h. Cells were analyzed using flow cytometry and used in a murine acute inflammation model. B. - E. Flow cytometry measured the binding of the following reagents to mouse neutrophils (CD11b+, Gr-1/Ly-6G/1A8+, F4/80-): B. VVA-FITC, C. Anti-mouse PSGL-1 mAb 2PH1, D. P-selectin-IgG and E. L-selectin-IgG. Ac 5 GalNTGc increased VVA-lectin binding by 4-5 fold, and reduced L-/P-selectin IgG binding by 50-70% without affecting PSGL-1 expression. F. mBMCs cultured with 80μM Ac 5 GalNTGc for 40h were mixed with VC at 1:1 ratio. In mix 1, Ac 5 GalNTGc cells were labeled with CMTMR (Red) while VC was CMFDA (Green) labeled. Labels were swapped in Mix 2 (dot plot not shown). Mix 1 or 2 cells were tail-vein injected into recipient mice following thioglycollate injection i.p. Red:green ratio of Gr-1+ cells in the peritoneal lavage and bone marrow was measured at 20h. Ac 5 GalNTGc reduced neutrophil counts in peritoneum by 50% in both Mixes. G-I. Ac 5 GalNTGc (100mg/kg/day) or VC was injected daily into mice for 4 days prior to induction of peritonitis. Murine neutrophil (CD11b+, Gr-1/Ly-6G/1A8+, F4/80-) counts in the peritoneum were quantified at 16h. Neutrophil counts in peritoneal lavage was reduced by 65% in Ac 5 GalNTGc treated mice (panel H ). VVA binding was augmented in peritoneal neutrophils (panel I ). * P <0.05 with respect to all other treatments in each panel, except as indicated.

Article Snippet: In some cases, Ac 5 GalNTGc and vehicle control treated BMCs were differentially tagged with cell tracker dyes CMFDA (5-chloromethylfluorescein diacetate, green) or CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine, red) following manufacturer’s instructions (Setareh Biotech, Eugene, OR).

Techniques: Isolation, Cell Culture, Flow Cytometry, Binding Assay, Expressing, Labeling, Injection