Journal: International Journal of Nanomedicine

Article Title: Sequential Release of mRNA Complex and T Cells by a Double-Layered Implantable Scaffold for Combination Therapy of Head and Neck Squamous Cell Carcinoma

doi: 10.2147/IJN.S582078

Figure Lengend Snippet: Characteristics of DMP nanoparticles and the proliferation-inhibiting effect of the DMP-mBim complex in vitro. ( A ) Size distribution and zeta potential of DMP nanoparticles. ( B ) Transmission electron microscopy (TEM) photomicrographs of DMP stained with phosphotungstic acid solution (scale bar: 50 nm). ( C ) Cell viability MTT assay of DMP and PEI25K in 293T cells. ( D ) Hemolysis test of DMP nanoparticles, Triton X-100 was used as positive control, and normal saline was used as negative control (***P<0.001, ****P<0.0001). ( E ) Critical micelle concentration evaluation of DMP using pyrene as the fluorescent dye. ( F ) Murine Bim mRNA (mBim) obtained by the in vitro transcription method was detected by electrophoresis. ( G ) Gel-retardation assay of mRNA and DMP at different weight ratios. ( H ) RNase protection electrophoresis assay of the DMP-mRNA complex. ( I ) Transfection efficiency of DMP-EGFP in SCC-VII cells (scale bar: 100 μm). ( J ) Bim levels in SCC-VII cells after DMP-mBim complex transfection (***P<0.001). ( K ) MTT assay of SCC-VII cells after DMP-mBim transfection and representative images. The cell viability and inhibition rate were also calculated (scale bar: 100 μm, ****P<0.0001). ( L ) Proliferation-inhibition ability of the DMP-mBim complex on SCC-VII cells evaluated by clonogenic assay (****P<0.0001). ( M ) Apoptosis-inducing ability of DMP-mBim complex in SCC-VII cells using Annexin V/PI staining by flow cytometry (****P<0.0001). ( N ) Live/dead staining evaluation of SCC-VII cells treated with the DMP-mBim complex (scale bar: 100 μm, ****P<0.0001).

Article Snippet: Cells were dual-stained for the plasma membrane and tubulin using DiI (Cell Plasma Membrane Staining Kit; Beyotime, China) and Tubulin-Tracker Green (Live Cell Staining Kit; Beyotime, China).

Techniques: In Vitro, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Staining, MTT Assay, Positive Control, Saline, Negative Control, Concentration Assay, Electrophoresis, Electrophoretic Mobility Shift Assay, Transfection, Inhibition, Clonogenic Assay, Flow Cytometry

Journal: International Journal of Nanomedicine

Article Title: Sequential Release of mRNA Complex and T Cells by a Double-Layered Implantable Scaffold for Combination Therapy of Head and Neck Squamous Cell Carcinoma

doi: 10.2147/IJN.S582078

Figure Lengend Snippet: Transfection efficiency and treatment effect of DMP-mBim complex after release from scaffold. ( A and B ) Representative fluorescence images of SCC-VII cells transfected with supernatants of the outer layer of the scaffold containing the DMP-EGFP complex in short-term release (scale bar: 50 μm) and detection of efficiency by flow cytometry. ( C and D ) Representative fluorescence images of SCC-VII cells transfected with supernatants of the outer layer of the scaffold containing the DMP-EGFP complex in long-term release (scale bar: 100 μm) and the calculated transfection efficiency. ( E ) Cumulative RNA-release rate in the outer layer scaffold. ( F ) Bim levels in SCC-VII cells after treatment with supernatants of the outer layer of the scaffold containing DMP-mBim complex (***P<0.001). ( G and H ) MTT assay of SCC-VII cells after treatment with the supernatants of the outer layer of the scaffold containing DMP-mBim complex (scale bar: 100 μm, ****P<0.0001, **P<0.01). ( I ) Live/dead staining evaluation of SCC-VII cells treated with the supernatants of the outer layer of the scaffold (scale bar: 100 μm, ****P<0.0001).

Article Snippet: Cells were dual-stained for the plasma membrane and tubulin using DiI (Cell Plasma Membrane Staining Kit; Beyotime, China) and Tubulin-Tracker Green (Live Cell Staining Kit; Beyotime, China).

Techniques: Transfection, Fluorescence, Flow Cytometry, MTT Assay, Staining

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