Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: DOCK2 maintains colonic IFN-γ-producing homeostatic T cells, impairs IL-22 and Reg3 AMP expression, and facilitates Verrucomicrobia colonization under physiological condition. ( A - G ) WT and Dock2 –/– mice were sacrificed under physiological conditions. Gross colonic appearance ( A ) and colon length measurements ( B ) were conducted. Following the isolation of leukocytes from the cLP, the proportions of CD4 + T cells (CD3ε + CD4 + ), CD8 + T cells (CD3ε + CD8α + ), γδT cells (CD3ε + TCRγδ + ), B cells (CD19 + ), NK cells (TCRγδ – NK1.1 + ), neutrophils (Ly6G + CD11b + ), monocytes (Ly6C + CD11b + ) and dendritic cells (MHC-II + CD11c + ) in CD45 + cells ( C ), the proportions of IFN-γ- or IL-17-producing cells in whole CD4 + and CD8 + T cells ( D ), and absolute numbers of IFN-γ- or IL-17-producing CD8 + T cells ( E ) were determined by FACS. The mRNA expression levels of genes encoding cytokines and T cell-associated transcription factors ( F ), and indicated AMPs and tight-junction proteins ( G ) in colon tissues were assessed by qPCR (n = 4/group) ( H , I ) mRNA expression levels of Muc2 (H) and Il18 ( I ) in colon tissues from WT and Dock2 –/– mice under normal or colitis conditions were examined by qPCR (n = 4/group). ( J ) Bacterial DNA was extracted from feces isolated from WT and Dock2 –/– mice under physiological conditions, and relative content of each indicated bacterial phylum or order was determined by qPCR using specific primers (WT, n = 11; Dock2 –/– , n = 5). ( K - M ) WT and Rorc –/– mice were sacrificed under physiological conditions. Following the isolation of leukocytes from the cLP, the proportions of IL-22/IL-17-producing cells in CD90.2 + Lineage – ILCs and in CD90.2 – Lineage + cells ( K ), absolute numbers of IL-22 + IL-17 + ILCs and IL-22 + IL-17 + Lineage + cells ( L ) were determined by FACS (n = 8/group). The mRNA expression levels of genes encoding IL-22, Reg3γ, Reg3β, and Mucin 2 in colon tissues were assessed by qPCR ( M ) (n = 6/group). ( N ) Bacterial DNA was extracted from feces isolated from WT and Rorc –/– mice under physiological conditions, and relative content of Akkermansia muciniphila was determined by qPCR using specific primers (n = 6/group). Data in ( A - N ) are from one of two independent experiments. Data in ( B - E , H - N ) are shown as mean ± SD and in (B-N) were analyzed using unpaired two-tailed Student’s t-test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Isolation, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: Dock2 signaling in T cells modifies IFN-γ- and IL-22-producing lymphocytes and protects mouse colitis in a commensal microbiota-dependent manner. ( A , B ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were sacrificed under physiological conditions (n = 4/group). Gross colonic appearance and colon length measurements ( A ) were conducted, and the mRNA expression of Reg3 family AMPs in colon tissues ( B ) were determined by qPCR. ( C - M ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were administered 2% DSS in drinking water for 7 days. Body weight loss ( C ) and disease activity index ( D ) were monitored daily during colitis induction (n = 3/group). On day 10 post-DSS treatment, mice were sacrificed, and gross colon observations, colon length measurements ( E , Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 7), and histological analyses of the distal colon (H&E staining) (F, G, n = 3/group) were conducted. Following leukocyte isolation from the cLP, the proportions of CD4 + and CD8 + T cells in leukocytes (H, Dock2 fl/fl n = 9, Cd4 -cre; Dock2 fl/fl n = 6), IFN-γ + in CD4 + and CD8 + T cells (I, Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 3) and IL-22 + and IL17 + populations in CD90.2 + Lineage – ILCs ( J ) and Lineage + lymphocytes ( K , Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 5) were quantified using FACS. The mRNA expression levels of genes encoding IL-22, RORγt and AHR in CD90.2 + Lineage – sorted ILCs ( L , Dock2 fl/fl n = 3, Cd4 -cre; Dock2 fl/fl n = 5) and the mRNA expression of Ifng , Il22 and Reg3 family AMPs in colon tissues ( M , Dock2 fl/fl n = 7, Cd4 -cre; Dock2 fl/fl n = 4) were determined by qPCR. ( N - S ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were co-housed for 4 weeks and then administered 2% DSS in drinking water for 7 days. Body weight loss ( N ) and disease activity index ( O ) were monitored daily during colitis induction. On day 10 post-DSS treatment, mice were sacrificed, and gross colon observations and colon length measurements ( P ), and histological analyses of the distal colon ( Q , H & E staining) were conducted (n = 3/group). Following leukocyte isolation from the cLP, the proportions of whole CD4 + and CD8 + T cells in CD45 + leukocytes (R) and IFN-γ + in CD4 + and CD8 + T cells (S) were quantified using FACS (n = 5/group) Data in ( A - B ) are from one of two, in ( C - M ) are from one of four, and in (N-S) are from one of two independent experiments. Data in ( B - D , F - S ) are shown as mean ± SD. Data in (B-D, F-S) were analyzed using unpaired two-tailed Student’s t-test. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Activity Assay, Staining, Isolation, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: Reg3 AMPs inhibit A. muciniphila expansion, and DOCK2-IFN-γ axis suppresses IL-22 by impairing Ahr transcription. ( A ) WT and Il22 –/– mice were sacrificed under physiological conditions, and the mRNA expression levels of genes encoding Reg3γ, Reg3β, and ZO1 in colon tissues were assessed by qPCR (WT, n = 5; Il22 –/– , n = 4). ( B ) A. muciniphila was cultured at 37 °C under anaerobic conditions with recombinant Reg3γ or Reg3β (5 μg/ml each peptide) added to the medium. Bacterial growth was measured spectrophotometrically at 3, 6, 9 and 12 h (n = 3 replicates/group). ( C ) WT mice were intrarectally administered recombinant Reg3γ or Reg3β for 5 h, followed by determination of A. muciniphila content in feces via qPCR (n = 4). ( D ) CD8 + or CD4 + T cells were isolated from splenocytes of WT mice, and were stimulated with anti-CD3/CD28 antibodies with the addition of DOCK2-Rac1 interaction inhibitor CPYPP. Four days after the stimulation, IFN-γ + in CD8 + or CD4 + T cells were quantified using FACS (n = 5 replicates/group). ( E ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AhR, and RORγt in ILCs were assessed by qPCR (n = 6 replicates/group). ( F ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ, AHR inhibitor BAY218 or AHR agonist ITE. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AHR, and RORγt in ILCs were assessed by qPCR (n = 3 replicates/group). ( G ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ and STAT1 antagonist fludarabine. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AHR, and RORγt in ILCs were assessed by qPCR (n = 3 replicates/group). ( H ) Logo plot of the predicted binding motif of the transcription factor STAT1 to the AhR promoter. ( I ) The efficiency of STAT1 overexpression was confirmed by immunoblot analysis. ( J ) AHR mRNA levels in HT29 cells overexpressing STAT1 or empty vector control were quantified by qPCR (n = 3 replicates/group). ( K ) Schematic of AHR promoter regions (P1–P5) with predicted STAT1-binding sites. STAT1 binding was assessed by ChIP using anti-Flag antibody as a bait from paraformaldehyde cross-linked HT29 cells, followed by RT–PCR. ( L ) AHR mRNA levels in HT29 cells treated with IFN-γ (5, 20 or 50 ng/ml) for 48 h were assessed by RT-qPCR (n = 3 replicates/group). ( M ) Luciferase activity driven by AHR promoter was examined in HT29 cells co-transfected with Renilla and pGL3-AHR reporter plasmids, with or without 20 ng/ml IFN-γ (n = 3 replicates/group). ( N ) H3K27me3 protein levels in HT29 cells treated with LPS or IFN-γ were analyzed by immunoblot. ( O ) AHR mRNA levels in HT29 cells treated with IFN-γ in the presence or absence of GSK126 were quantified by qPCR (n = 3 replicates/group). ( P ) H3K27me3 protein levels in HT29 cells treated with increasing concentrations of GSK126 were analyzed by immunoblot. ( Q ) AHR mRNA levels in HT29 cells exposed to graded concentrations of GSK126 were assessed by qPCR (n = 3 replicates/group). ( R ) H3K27me3 enrichment at the AHR promoter following IFN-γ treatment for 24 h in HT29 cells were assessed by ChIP coupled with RT–qPCR (n = 3 replicates/group). ( S ) H3K27me3 enrichment at the AHR promoter following IFN-γ treatment with indicated doses in HT29 cells were assessed by ChIP coupled with RT–qPCR (n = 3 replicates/group). ( T - V ) WT mice were intraperitoneally administered AhR inhibitor BAY-218 (10 mg/kg) every other day for 3 times. On day 6 after the first administration, feces were harvested and A. muciniphila content was determined via qPCR ( T ) (n = 6). The mRNA expression levels of Ahr and Il22 in ILCs ( U ) (n = 3) and the expression of Reg3g , Reg3b , Ahr and Il22 in colon tissues ( V ) (non-treated n = 6, BAY-218 n = 4) were assessed by qPCR. Data in ( B ) are from one of three and in ( A , C - G , I - V ) are from one of two independent experiments. Data in ( A , B , D - G , J , L , M , O , Q – V ) are shown as mean ± SD. Data in ( A , J , M , T - V ) were analyzed using unpaired two-tailed Student’s t-test, in ( C ) using paired two-tailed Student’s t-test, and in ( B , D - G , L , O , Q - S ) using one-way ANOVA followed by Tukey’s multiple-comparisons test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Cell Culture, Recombinant, Isolation, Binding Assay, Over Expression, Western Blot, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: DOCK2-IFN-γ axis is also in the development of human IBD. ( A ) Colon tissues from one CD patient were obtained after resection, and immunohistochemical (IHC) staining for DOCK2 and REG3γ proteins of inflammatory colon tissues was performed. ( B ) Colon tissues from CD patients and healthy individuals were collected, and the relative mRNA expression of DOCK2 , IFNG , and REG3G in ileum and rectum tissues was determined by bulk RNA-seq analysis (HC, n = 44; CD, n = 46). ( C ) Point plots show the correlation of relative mRNA expression between DOCK2 and IFNG , and between AHR and IL22 by bulk RNA-seq analysis as described in ( B ). ( D ) CD3 + T cells were isolated from the inflammatory colon tissue after resection, and were stimulated with anti-CD3/CD28 antibodies with the addition of DOCK2-Rac1 interaction inhibitor CPYPP. Four days after the stimulation, the mRNA expression level of IFNG was assessed by qPCR (n = 4 replicates/group). ( E – G ) One public scRNA-seq dataset of colon cells obtained from IBD patients ( GSE150516 ) was reanalyzed. Heatmap shows the expression levels of top typical genes in each identified DOCK2 + cell cluster (subset) and Umap shows the cell types of DOCK2 + colonic cells ( E ). Violin plots display the expression of indicated genes in each identified cell cluster ( F ). The point plots show the correlation between the expression levels of DOCK2 and IFNG , and between AHR and IL22 in double-positive CD8 + or CD4 + T cells ( G ). ( H ) One public scRNA-seq dataset of colon cells obtained from IBD patients ( GSE214695 ) were reanalyzed, and the point plots show the correlation between the expression levels of DOCK2 and IFNG in double-positive CD8 + T cells in pooled colonic cells from 6 UC and 6 CD patients. ( I ) Point plots show the correlation between the expression levels of DOCK2 and CD8A , DOCK2 and IFNG , REG3G and IL22 , and AHR and IL22 in the colon tissues from all pooled human samples in the public RNA-seq dataset ( GSE117993 ). ( J ) The relative abundance of A. muciniphila in fecal bacteria from IBD patients and healthy individuals was determined by reanalyzing a public metagenomics sequencing dataset ( GSE111889 , HC n = 429, UC n = 459, CD n = 750). ( K ) The relative abundance of A. muciniphila in fecal bacteria from CD patients and healthy individuals was determined by 16S rDNA sequencing analysis (HC, n = 77; CD, n = 80). ( L ) The transcriptional level of the REG3G gene in colon tissues from non-IBD controls and IBD patients was determined by reanalyzing a public bulk RNA-seq dataset ( GSE117993 , Not IBD n = 55, IBD n = 135). ( M ) Point plots show the correlation between the relative abundance of A. muciniphila and the expression levels of REG3G , REG3A or IFNG in rectum tissues from the same individual UC patients in the public RNA-seq dataset ( GSE111889 ). Data in ( A , D ) are from one of two independent experiments. Data in ( B , J - L ) are shown as mean ± SEM, and in ( D ) are shown as mean ± SD. Data in ( B , D , J - L ) were analyzed using unpaired two-tailed Student’s t-test. Correlation significance in panels ( C , G - I , M ) was determined using Pearson’s correlation coefficient (r 2 ) test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, RNA Sequencing, Isolation, Bacteria, Metagenomics, Sequencing, Two Tailed Test

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 5 | ISL1 is a critical regulator of human motoneuron development. (a) Immunofluorescence of ISL1 (red) in cells after 15-day differentiation from H1 hESCs. Cell nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole; blue). RA-D1, RA-D3 and RA-D6 denote the time of initiation of RA patterning (at day 1, day 3 and day 6, respectively). (b) Percentage of ISL1-positive cells under conditions described in a. Each bar represents mean±s.d. (error bars) of six experiments. (c) Fluorescence images of ISL1 (left), ISL1/DAPI (middle) and ISL1/ TUJ1 (right) staining in differentiated cells replated at a low density for 3 days after 15-day differentiation. (d) Fluorescence images of differentiated hESCs with NT shRNA (‘Control’, left) or with ISL1 shRNA-1 (‘ISL shRNA-1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (e) Fluorescence images of differentiated hESCs with NTshRNA (‘Control’, left), ISL1 shRNA-2 (‘ISL shRNA-2’, middle) and ISL1 shRNA-2 with ectopic ISL1 expression (‘shRNA-2 þ ISL1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (f) Fluorescence images of differentiated hESCs with NTshRNA (left) or with ISL1 shRNA (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation. Cell nuclei were stained with DAPI (blue). (g) Fluorescence images of differentiated hESCs with NT shRNA (left), ISL1 shRNA-2 (middle) and ISL1 shRNA-2, with ectopic ISL1 expression (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). All scale bars, 50 mm.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Staining, Fluorescence, shRNA, Control, Expressing

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 6 | Summary of the MN differentiation model and the stages of neural differentiation. (a) hESCs undergoing neural induction begin to express high levels of NPC markers, including PAX6 and SOX1 at day 3, while markedly downregulating the pluripotency markers such as OCT-4 and NANOG (albeit retaining some degree of expression). Thus, the cells at day 3 might represent a population of primitive NPCs that can be induced to become anterior NPCs and also possesses the potential to differentiate posteriorly. With neural patterning initiated at day 3 after neural induction, the early MN marker ISL1 can be seen at day 13 after hESC differentiation, while mature MN markers including HB9 and ChAT are expressed after 17-day differentiation. (b) Our results point to a previously unidentified primitive stage of neural progenitors that is intermediate between pluripotent hESCs and anterior NPCs. This population of potential primitive NPCs can be induced to become anterior NPCs in the absence of patterning factors, and also possesses the potential to differentiate along the posterior fate in the presence of patterning factors.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Expressing, Marker

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: RAW264.7 cells were stimulated with mEVs and treated with recombinant mouse Reg3g protein (100 ng/ml, #HY- P79024 , MedChemExpress) or not for 24 h. The corresponding cultured cells were lysed in RIPA buffer (#P0013, Beyotime) supplemented with a protease inhibitor cocktail (#P1048, Beyotime).

Techniques: Derivative Assay, Flow Cytometry, Transfection, Staining, Labeling, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, 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: 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: RAW264.7 cells were stimulated with mEVs and treated with recombinant mouse Reg3g protein (100 ng/ml, #HY- P79024 , MedChemExpress) or not for 24 h. The corresponding cultured cells were lysed in RIPA buffer (#P0013, Beyotime) supplemented with a protease inhibitor cocktail (#P1048, Beyotime).

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: RAW264.7 cells were stimulated with mEVs and treated with recombinant mouse Reg3g protein (100 ng/ml, #HY- P79024 , MedChemExpress) or not for 24 h. The corresponding cultured cells were lysed in RIPA buffer (#P0013, Beyotime) supplemented with a protease inhibitor cocktail (#P1048, Beyotime).

Techniques: Immunofluorescence, Western Blot, Binding Assay, ChIP-qPCR, Fluorescence, Immunoprecipitation, 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 deficiency aggravates HFD-induced pancreatic islet inflammation and increases proinflammatory macrophages expansion in islets. (A) Schematic diagram of animal experiments (n = 6). (B) Blood glucose levels during the oral glucose tolerance test (OGTT), and the AUC of OGTT in WT and Reg3g −/− mice fed with CD or HFD for 13 weeks. (C) Fast blood glucose levels of the mice fed with CD or HFD for 14 weeks. (D) Glucose stimulated insulin secretion in WT and Reg3g −/− mice fed with CD or HFD for 13 weeks. (E) Immunostaining and quantification of islet β cell (insulin + ) and α cell (glucagon + ) in pancreatic sections of WT and Reg3g −/− mice fed with CD or HFD for 14 weeks. Scale bar, 200 μm. (F) The representative FACS plots and percentage of CD11b + F4/80 + cells harvested from pancreas of WT and Reg3g −/− mice fed with CD or HFD for 14 weeks. (G) The expression of CD11c analyzed under CD11b + F4/80 + gated in pancreas. (H) The expression of CD206 analyzed under CD11b + F4/80 + gated in pancreas. (I) Immunostaining of CD11c + (red) and CD206 + (green) in pancreatic sections of WT and Reg3g −/− mice fed with CD or HFD. Scale bar, 20 μm. (J) Schematic diagram of animal experiments (n = 6). (K) 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. Data are represented as means ± SEMs. ∗ P < 0.05, ∗∗∗ P < 0.001 by Student's unpaired two-tailed t -test.

Article Snippet: RAW264.7 cells were stimulated with mEVs and treated with recombinant mouse Reg3g protein (100 ng/ml, #HY- P79024 , MedChemExpress) or not for 24 h. The corresponding cultured cells were lysed in RIPA buffer (#P0013, Beyotime) supplemented with a protease inhibitor cocktail (#P1048, Beyotime).

Techniques: Immunostaining, Expressing, Flow Cytometry, Labeling, Isolation, Fluorescence, Two Tailed Test

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 improves glucose homeostasis and restricts inflammation in pancreatic islet of T2DM model mice. (A) Schematic diagram of animal experiments (n = 10). (B) Statistics on the incidence of diabetes after STZ injection. (C) Fast blood glucose levels of the mice at indicated time point. (D) Blood glucose levels during the OGTT, and the AUC of OGTT at 13th week. (E) Blood glucose levels during the ITT, and the AUC of ITT at 13th week. (F) Glucose stimulated insulin secretion at 13th week. (G) Schematic diagram of animal experiments (n = 6). (H) Western blot analysis of Reg3g in pancreas. (I) Fast blood glucose levels of the mice at indicated time point. (J) Blood glucose levels during the OGTT, and the AUC of OGTT at 13th week. (K) Glucose stimulated insulin secretion at 13th week. (L) Immunostaining and quantification of islet β cell (insulin + ) and α cell (glucagon + ) in pancreatic sections. Scale bar, 200 μm. (M) The representative FACS plots and percentage of CD11b + F4/80 + cells harvested from pancreas. (N) The expression of CD11c analyzed under CD11b + F4/80 + gated in pancreas. (O) The expression of CD206 analyzed under CD11b + F4/80 + gated in pancreas. (P) Immunostaining of CD11c + (red) and CD206 + (green) in pancreatic sections. Scale bar, 20 μm. (Q) Schematic diagram of animal experiments (n = 6). (R) 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. Data are represented as means ± SEMs. ∗ P < 0.05, ∗∗∗ P < 0.001 by Student's unpaired two-tailed t -test.

Article Snippet: RAW264.7 cells were stimulated with mEVs and treated with recombinant mouse Reg3g protein (100 ng/ml, #HY- P79024 , MedChemExpress) or not for 24 h. The corresponding cultured cells were lysed in RIPA buffer (#P0013, Beyotime) supplemented with a protease inhibitor cocktail (#P1048, Beyotime).

Techniques: Injection, Western Blot, Immunostaining, Expressing, Flow Cytometry, Labeling, Isolation, Fluorescence, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1186/s13024-025-00840-1

Figure Lengend Snippet: AD risk SNP rs1532278 shows allele-specific open chromatin (ASoC) and cis-regulates CLU expression in iGlut. A Schematic research design. ATAC-seq was performed to identify OCRs and GWAS risk SNPs that showed ASoC in iPSC Glutaminergic (iGlut), GABAergic (iGABA), and dopaminergic (iDA) neurons, and microglia (iMG) and astrocytes (iAst), which is followed by transcription factor (TF) binding prediction to confirm putative functional AD risk SNP, CRIPSR-cas9 SNP editing, and functional assays in cells. B ASoC mapping identifies intronic rs1532278 as a putatively functional SNP among several other GWAS risk SNPs equivalently associated with AD at the CLU locus. Note that rs1532278 is the only SNP within the neuronal OCR with a stronger OCR peak in iGlut. C T and C alleles of rs1532278 show differential allelic ATAC-seq reads (i.e., ASoC) in iGlut. The bottom panel shows the two most conserved TF binding motifs at the SNP site. D Schematic CLU gene structure near rs1532278 (upper panel) and a diagram showing CRISPR-Cas9 editing of rs1532278 in two iPSC lines (CD05 and CD07; T/C) to covert T/C lines to isogenic T/T and C/C lines (middle panel, representative Sanger sequencing result from CD07 line). Representative images of iGlut (CD07 line) of all three genotypes are also shown (bottom panel); MAP2 and HuNu (human nuclear antigen) staining shows the morphology of iGlut and neuron purity in iGlut-mAst co-cultures. E – F ISL2 ChIP-qPCR on day 30 iGlut-mAst co-cultures. n = 3 biological replicates from one clone per line of one independent differentiation. G - H ISL2 siRNA knockdown in day- 30 pure iGlut (T/T) cultures. Samples of 72 h post-siRNA transfection were used for qPCR. n = 3 biological replicates from one clone per line of one independent differentiation. I Neuronal CLU mRNA level in isogenic iGlut-mAst co-cultures of different genotype of rs1532278 (human-specific CLU qPCR assay was used). n = 6 biological replicates per group (2–3 clones per line and 2–3 biological replicates for each clone) from two independent differentiations of each line. J Secreted CLU (sCLU) detected by ELISA from the supernatant of iGlut-mAst co-cultures. n = 4 biological replicates per group (2 clones per line and 2 biological replicates per clone) from two independent differentiations of each line. K - L Immunofluorescence staining of CLU in day 25 pure iGlut cultures. n = 6–7 coverslips per group (In total: 2 clones per line and 3–4 coverslips for each clone and 3–5 images per coverslip; shown are example images of CD05) from two independent differentiations of each line. Data, mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Scale bars are indicated in each image

Article Snippet: Cells were harvested in ice-cold PBS, pelleted, sonicated, and immunoprecipitated with 10 μg ISL2 (R and D Systems, AF4244, Sheep) or DRGX antibodies (Bioss, bs- 11827R, Rabbit) and Protein A/G magnetic beads overnight at 4 °C.

Techniques: Expressing, Binding Assay, Functional Assay, CRISPR, Sequencing, Staining, ChIP-qPCR, Knockdown, Transfection, Clone Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence