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: Rewiring of macrophage induced by Reg3g relies on P2RX7 downregulation. (A–D) The RAW264.7 cells were treated with Reg3g or PBS for 24 h in the presence of mEVs in these indicated experiments. (A) mRNA levels of P2rx7 were determined by RT-qPCR in RAW264.7 (n = 5). (B–C) Western blot and quantification analysis of P2RX7 in whole cell lysate (n = 3). (D) Immunofluorescent staining of P2RX7 in mito-tracker Red labeled RAW264.7 (n = 3). (E–J) The BMDM cells were treated with A438079 and Reg3g for 24 h in the presence of mEVs in these indicated experiments. (E) Flow cytometry analysis of CD11c + and CD206 + cells ratios (n = 5). (F) mRNA levels of Il1b, Cd11c, Tnfα, iNos, Cd206, and Retnla were determined by RT-qPCR (n = 5). (G) Phagocytosis detection using FITC-Dextran (n = 5). (H) ECAR analysis in BMDMs (n = 6). (I) OCR analysis in BMDMs (n = 6). (J) Contents of lactate detection (n = 5). Data are presented as the means ± SEMs. For A and C, statistical significance was calculated using Student's unpaired two-tailed t -test. For E, F, G, H, I and J, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ 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: Quantitative RT-PCR, Western Blot, Staining, Labeling, Flow Cytometry, 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 downregulates P2RX7 by promoting HSPG-NF-κB complex formation to maintain NF-κB in the cytoplasm. (A–C) The RAW264.7 cells were treated with Reg3g or PBS for 24 h in the presence of mEVs in these indicated experiments. (A) Representative NF-κB immunofluorescence (n = 3). (B) Western blot analysis of NF-κB in whole cell lysate (n = 3). (C) Western blot analysis of NF-κB in cell nucleus lysate (n = 3). (D) Predicted NF-κB binding site motif. (E) The putative NF-κB binding sites in P2rx7 promoter. The start site of transcription (TSS) is denoted +1 and the location of potential NF-κB binding site has been shown relative to TSS. (F–H) The RAW264.7 cells were treated with Reg3g in the presence of mEVs in these indicated experiments. (F) ChIP-qPCR analyzed the binding of NF-κB to P2rx7 promoter (n = 3 to 5). (G) Co-localization of HSPG and NF-κB. The line charts represent fluorescence intensity (MFI), which is presented the distance from α to ω in the images (n = 3). (H) The interaction of HSPG and NF-κB was determined by co-immunoprecipitation (n = 3). Data are presented as the means ± SEMs. For B and C, statistical significance was calculated using Student's unpaired two-tailed t -test. For F, statistical significance was calculated using ANOVA with Tukey's post hoc comparison. ∗ P < 0.05, ∗∗ 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: Immunofluorescence, Western Blot, Binding Assay, ChIP-qPCR, Fluorescence, Immunoprecipitation, Two Tailed Test, Comparison