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: Frontiers in Immunology

Article Title: Gemcitabine-cisplatin chemotherapy plus anti-PD-L1 therapy reinvigorates antitumor immune response by reprogramming the intrahepatic cholangiocarcinoma microenvironment

doi: 10.3389/fimmu.2025.1666393

Figure Lengend Snippet: Differential analysis of CD8 + T cells between untreated and GCP-treated ICC samples. (A) UMAP plots showing all T cells labeled in different colors according to cell type annotation. (B) Volcano plot showing the differentially expressed genes of intra-tumoral CD8 + T cells between pretreated and posttreated ICC samples. (C) Immunofluorescence images showing the infiltration of CD8 + CD81 + T cells in treatment-naïve ICC samples from the TMA cohort (n=89). Kaplan–Meier survival curves for OS (D) and RFS (E) of 89 ICC patients grouped by infiltration levels of CD8 + CD81 + T cells. P values were determined via log-rank test. (F) Boxplot showing infiltration levels of CD8 + CD81 + T cells in GCP-treated tumors revealed by multiplex immunofluorescence in GCP cohort. (G) Violin plots showing the expression scores of tissue resident, exhausted, and co-stimulatory gene signatures in tumor-infiltrating CD8 + T cells between pretreated and posttreated samples. The P values were calculated by Wilcox test. OS, overall survival; RFS, recurrence-free survival. ****p < 0.0001.

Article Snippet: After that, the slides were incubated with CD81 primary antibody (1:200 dilution, A01281-2, BOSTER) and Alexa Fluor 488 donkey anti-rabbit antibody as described above.

Techniques: Labeling, Immunofluorescence, Multiplex Assay, Expressing