Journal: Redox Biology

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

doi: 10.1016/j.redox.2025.103996

Figure Lengend Snippet: Macrophages ingest β cell-derived dysfunctional mitochondria in the form of extracellular vesicles. (A) Generation of mtDsRed2-labeled MIN6 cells, cocultured with DIO-labeled BMDM for indicated time. (B) Representative confocal images of BMDM incubated with MIN6 cells (described in A) for 0.5, 12, and 24 h (n = 3). (C) Time-lapse confocal imaging revealing a mitochondria uptake event in macrophage. (D) qPCR analysis using specific primers for mtDNA from RAW264.7 and β-TC6 (n = 3). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 individual cultured (Mon-RAW264.7) or cocultured with β-TC6 (Co-RAW264.7) (n = 3). (F) Flow cytometry measured the transfer of mitochondria from Ins2p-mMito-DsRed2-labeled β cells to CD11b + F4/80 + macrophages isolated from mice islets and quantified the mean fluorescence intensity of mtDsRed2 (n = 6). (G) Representative confocal images of insulin immunofluorescence staining of mtDsRed2-labeled isolated islets (left). Flow cytometry measured mtDsRed2 in BMDM from the transwell system co-cultured with or without mtDsRed2-labeled isolated islets for 24 h (right) (n = 5). (H) The coculture and transwell culture systems determine the form of mitochondrial transfer. The mean fluorescence intensity of mtDsRed2 in RAW264.7 was analyzed by flow cytometry (n = 5). (I) Experimental schematic for collecting extracellular vesicles (EVs) from β cell-conditioned medium. (J) Proteomic analysis of mEVs. The six most representative cellular components are shown. Data are obtained from a pool of mEVs derived from β cells. (K) Representative confocal microscopy pictures of EVs from mtDsRed2-labeled β cells stained with DIO (n = 3). (L) Representative transmission electron microscopy image of EVs. The red arrow shows mitochondria in the EVs (n = 3). (M) Representative Western blot of Large EVs filtrated with 0.22 μm pore-size filter or not. CD63 and CD81 were used as markers of EVs, and TOM20 stands for mitochondria (n = 3). (N) Mitochondria membrane potential detection of β cells treated with or without PA and EVs from these β cells (n = 3). (O) Mitochondrial ROS content of EVs, from β cells treated with PA or not, was analyzed by MitoSOX staining (n = 6). Data are presented as the means ± SEMs. For D, E, F, G and O, statistical significance was calculated using Student's unpaired two-tailed t -test. For N, statistical significance was calculated using one-way ANOVA with Tukey's post hoc comparison. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The mouse pancreatic β cell lines MIN6 (RRID:CVCL_0431) and β-TC6 (RRID:CVCL_0605) were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China) and maintained in DMEM containing 10 % (for MIN6)/15 % (for β-TC6) FBS, and 1 % penicillin-streptomycin.

Techniques: Derivative Assay, Labeling, Incubation, Imaging, Cell Culture, Flow Cytometry, Isolation, Fluorescence, Immunofluorescence, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Pore Size, Membrane, Two Tailed Test, Comparison

Journal: Redox Biology

Article Title: Islet regeneration protein Reg3g promotes macrophage clearance of β cell-derived dysfunctional mitochondria-rich vesicles to mitigate T2DM

doi: 10.1016/j.redox.2025.103996

Figure Lengend Snippet: Reg3g facilitates macrophage to clear β cell-derived mitochondria by mitophagy, lowering proinflammatory responses. (A) Contents of Heparan sulfate detection in RAW264.7 cells treated with Reg3g or not for 24 h in the presence of mEVs (n = 6). (B) Flow cytometry analysis of MIN6-derived mtDsRed2 in RAW264.7 treated with Reg3g or not in the presence of mEVs (n = 5). (C) qPCR analysis of mtDNA from β-TC6 in RAW264.7 treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (D) Flow cytometry analysis of β cell-derived mtDsRed2 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g or not in the presence of mEVs (n = 5). (E) qPCR analysis of mtDNA from β-TC6 in RAW264.7 after transfected with siExtl3 or mimic and treated with Reg3g under β-TC6-derived mEVs-stimulated condition (n = 3). (F–J) The RAW264.7 cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (F) Immunofluorescent staining of LC3 + autophagosomes and co-localization with mtDsRed2 + in RAW264.7 (n = 3). (G) Co-localization of exogenous mtDsRed2 + mitochondria and Lyso-Tracker-labeled lysosome within RAW264.7 (n = 3). (H) Representative transmission electron microscopy image of RAW264.7. The red arrow shows mitophagy (n = 3). (I) Western blot analysis of mitophagy-related protein in RAW264.7 (n = 3). (J) RAW264.7 cells were transfected with AdPlus-mCherry-GFP-LC3B to detect the autophagosome formation (n = 3). (K–P) The BMDM cells were treated with Reg3g or not in the presence of mEVs in these indicated experiments. (K) Flow cytometry analysis of CD11c + and CD206 + cells ratios (n = 5). (L) mRNA levels of Il1b, Cd11c, Tnfα, iNos, Cd206, Ym1, Arg1 and Retnla were determined by RT-qPCR (n = 5). (M) Phagocytosis detection using FITC-Dextran (n = 5). (N) Contents of lactate detection (n = 5). (O) Extracellular acidification rate (ECAR) analysis in BMDMs (n = 6). (P) Oxygen consumption rate (OCR) analysis in BMDMs (n = 6). Data are presented as the means ± SEMs. For A, B, C, I, K, L, M, N, O and P, statistical significance was calculated using Student's unpaired two-tailed t -test. For D and E, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The mouse pancreatic β cell lines MIN6 (RRID:CVCL_0431) and β-TC6 (RRID:CVCL_0605) were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China) and maintained in DMEM containing 10 % (for MIN6)/15 % (for β-TC6) FBS, and 1 % penicillin-streptomycin.

Techniques: Derivative Assay, Flow Cytometry, Transfection, Staining, Labeling, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Two Tailed Test, Comparison