Journal: International Journal of Molecular Medicine

Article Title: ZEB1 maintains mitochondrial fission and macrophage efferocytosis by restraining MFN2, thereby limiting inflammation and improving tendon-bone healing

doi: 10.3892/ijmm.2026.5805

Figure Lengend Snippet: ZEB1 knockdown affects the cell efferocytosis efficiency of macrophages. (A) After 6 days of induction culture with M-CSF, the expression level of F4/80 (green) was detected by immunofluorescence staining (scale bar, 20 µ m). (B) Flow cytometry was performed to detect the proportion of F4/80-positive cells to verify the purity of macrophages. (C) The mRNA expression levels of ZEB1 in macrophages at different time points (0, 1, 3 and 6 h) of co-culture were determined by RT-qPCR. (D) After co-incubation of BMDMs with ACs for 0, 1, 3 and 6 h, the protein expression of ZEB1 was detected by western blotting and semi-quantitative analysis of ZEB1 expression was conducted. (E) The mRNA expressions of TNFα, iNOS, CD206 and Arg-1 were detected by RT-qPCR. (F) The expression of ZEB1 mRNA was detected by RT-qPCR to evaluate the knockout efficiency. (G) Detection of ZEB1 protein expression by WB and semi-quantitative analysis of ZEB1 expression. (H) BMDMs were labeled with Mito-Tracker (green label), and then co-cultured with PI-labeled apoptotic Jurkat cells at a ratio of 5:1 of apoptotic cells to macrophages for 6 h. After removing the non-phagocytic apoptotic cells, the proportion of PI-positive macrophages (indicated by arrows) among the total macrophages was determined by fluorescence microscopy (Scale bar, 20 µ m). (I) The apoptosis rate of cells induced by UV radiation was detected by flow cytometry (Annexin V-FITC/PI double staining) to prepare target cells for the efferocytosis assay. n=3. * P<0.05 vs. NC or 0 h groups. NC, negative control; sh, short hairpin; ZEB1, zinc finger E-box binding homeobox 1; BMDMs, bone marrow-derived macrophages; ACs, apoptotic cells; M-CSF, macrophage colony-stimulating factor; RT-qPCR, reverse transcription-quantitative PCR; iNOS, inducible nitric oxide synthase.

Article Snippet: The human lymphocyte line Jurkat (clone E6-1; American Type Culture Collection) was selected as the apoptotic cell model. Logarithmic growth phase Jurkat cells (1×10 6 cells/ml) were seeded in six-well plates and exposed to ultraviolet (UV) light under uncovered conditions.

Techniques: Knockdown, Expressing, Immunofluorescence, Staining, Flow Cytometry, Co-Culture Assay, Quantitative RT-PCR, Incubation, Western Blot, Knock-Out, Labeling, Cell Culture, Fluorescence, Microscopy, Double Staining, Negative Control, Binding Assay, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction