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: Antibodies used to determine protein expression list as follows: CD63 (Sc-5275, Santa), CD81 (HY– P80608 , MCE), LC3A/B (#12741, CST; #AF5402, Affinity Biosciences), TOM20 (66777-1-Ig, Proteintech), P2RX7 (28207-1-AP, Proteintech), NF-κB (#8242, CST), p–NF–κB (#3033, CST).

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: 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: Antibodies used to determine protein expression list as follows: CD63 (Sc-5275, Santa), CD81 (HY– P80608 , MCE), LC3A/B (#12741, CST; #AF5402, Affinity Biosciences), TOM20 (66777-1-Ig, Proteintech), P2RX7 (28207-1-AP, Proteintech), NF-κB (#8242, CST), p–NF–κB (#3033, CST).

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: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of generating engineered Tluc-EVs by introducing TlucCD9-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescence intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase in engineered Tluc-CD9 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD9EVs over PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of tetraspanins in EVs. H. Interaction network of CD9 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Fluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT, PanKO, CD9KO, CD63KO, and CD81KO cells expressing TlucCD63-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD63-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescent intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD63 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD63EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD63 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD81-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The MFI of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD81 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD81EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD81 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: ** p < 0.01,** * p < 0.001,**** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Generation of CD63-mNG-EVs in WT, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells. A. Schematic workflow of engineered mNG-EVs by introducing CD63-mNG lentiviruses “created with BioRender.com ”. B. Percentage of mNG positive cells after transduction using flow cytometry. C. MFI of the cells using flow cytometry. D. The flow cytometry plot for the cells after transduction, stained with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Quantification of engineered CD63-mNG EVs from 17 µL of CM collected from KO) and WT cells. F. Imaging flow cytometry plot for the mNG-EVs derived from stably expressing mNG cells. The data are presented as means (±SD, n = 2-3). One-way ANOVA was used to show significance and was illustrated as follows: * p< 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Flow Cytometry, Staining, Imaging, Derivative Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Proteomic evaluation on the restoration and expression of CD63 in EVs. (A) Expression of CD63 in EVs originating from CD63KO and WT HEK293T cells. The graph indicates the relative difference in CD63 level across samples using log2-transformed protein intensities centered across all samples. (B) Expression of CD63 in EVs from CD63KO cells transfected with Tluc-CD63 compared to CD63KO and WT cells. The graph indicates the log2 fold change in the relative CD63 level over CD63 KO EVs or WT EVs. Results represent data from three biological replicates.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Expressing, Transformation Assay, Transfection