Journal: bioRxiv

Article Title: Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

doi: 10.1101/2021.02.24.432630

Figure Lengend Snippet: (A) Representative immunofluorescent images show apoEV (labeled with CellMask™ Deep Red, red) surface expression of vesicle-localized-soluble N -ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs, green) VAMP3, V VAMP4 and Golgi SNARE of 15 kDa (GS15). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs. Scale bars = 25 μm. (B) Western blot analysis show expression of VAMPs in apoEVs compared to their source MSCs. (C) Western blot analysis show expression of VAMPs in apoEVs. Source MSCs were transfected with small interferon RNAs (siRNAs) as a negative control (siCtrl) for VAMP3 (si Vamp3 ), VAMP4 (si Vamp4 ), and both VAMP3 and VAMP4 (si Vamp3/4 ). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs after knockdown of VAMPs. (D) Representative images show cultured Fas mutant ( Fas mut ) tetraploid hepatocytes (4N-HCs) with 130 kDa Golgi matrix protein (GM130) immunofluorescent staining (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). White dashed lines depict cell borders of cultured HCs. Imaging analysis was performed to quantify apoEV uptake and percentages of Golgi-contacted apoEVs. Scale bars = 10 μm. N = 4 per group. (E) Representative fluorescent images show cultured Fas mut 4N-HCs with ac-α-tubulin immunostaining (white), N -acetylgalactosaminyltransferase 2 (GALNT2)-GFP-labeled Golgi (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). Imaging analysis was performed to quantify HC percentages with Golgi fragmentation and acetylated microtubules (MTs) fluorescence intensity. Scale bars = 5 μm. N = 4 per group. (F) Western blot analysis showed human VAMP3 (hVAMP3) expression in isolated HC Golgi. ApoEVs were derived from human MSCs (hApoEVs). GM130 was used as an internal control. (G) Western blot analysis showed VAMP3 expression in apoEVs without or with treatment by 250 ng/ml Tetanus toxin (TeNT). (H) Representative Golgin84 immunofluorescent staining (green) images of liver Golgi counterstained with DAPI (blue). WT, wild type. Imaging analyses were performed to quantify HC percentages with Golgi fragmentation. Scale bars = 5 μm. N = 4 per group. (I) Golgi apparatus was isolated from the liver and Golgi protein mass was determined using the BCA method. N = 4 per group. (J) Representative liver fluorescent images showed HCs with different nuclei (blue, DAPI for DNA) and their cell borders (green, phalloidin for F-actin). # indicates binucleated HCs. Scale bars = 25 μm. N = 4 per group. (K) After PI staining, percentages of binucleated HCs were quantified. Diploid HCs (2N-HCs) were analyzed using flow cytometry. N = 4 per group. (L) Hematoxylin and eosin (H&E) staining of liver tissues in periportal vein (PV) areas. Hepatic injury scores were examined based on pathological parameters. Scale bars = 50 μm. N = 4 per group. (M) Serum alanine aminotransferase (ALT) levels were determined. N = 4 per group. (N) Graphical summary illustrating that apoEVs use VAMP3 to assemble with Golgi for HC and liver regulation. Data represent mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, not significant, P > 0.05. Statistical analyses were performed by Student’s t test for two-group analysis or one-way analysis of variance followed by the Newman-Keuls post hoc tests for multiple group comparisons.

Article Snippet: For IF staining of surface markers of apoEVs, after isolation, apoEVs were stained with rabbit anti-human/mouse VAMP3 primary antibody (13640, Cell Signaling Technology, USA), rabbit anti-mouse VAMP4 primary antibody (PA1-768, Invitrogen, USA), mouse anti-mouse GS15 primary antibody (610960, BD Transduction Laboratories, UK), rabbit anti-mouse C1q primary antibody (ab71940, Abcam, UK), or mouse anti-mouse TSP1 primary antibody (sc-59887, Santa Cruz Biotechnology, USA) at 4°C for 1 h at a concentration of 1:100 in PBS.

Techniques: Labeling, Expressing, Staining, Western Blot, Transfection, Negative Control, Knockdown, Cell Culture, Mutagenesis, Imaging, Immunostaining, Fluorescence, Isolation, Derivative Assay, Control, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: 4-HNE reduces phagocytosis through the expression of Synaptotagmin 1 in human monocyte-derived macrophages

doi: 10.1101/2025.11.24.690103

Figure Lengend Snippet: Human peripheral blood monocyte-derived macrophages were transfected with SYT1-GFP (green) and treated with zymosan labeled with Alexa fluor 647 (white). A) Representative confocal images of a live cell (upper row) and a fixed cell with a phagosome (lower row). The orthogonal views are shown for the merge. The scale bar represents 5 microns. B) Representative confocal images of fixed cells expressing SYT1-GFP (green) and incubated with zymosan Alexa fluor 647 (white; only shown in the merge). The cells were labeled with an Alexa fluor 568 conjugated membrane probe, immunolabeling for lysosomal marker LAMP1 or endosomal marker VAMP3 (magenta). The scale bars indicate 5 microns, and the lines indicate the positions of the fluorescence intensity profiles (right panels). C) The Pearson correlation coefficients were determined for the membrane probe, LAMP1, and VAMP3 for 3 donors. Data are represented as median, and each data point represents one cell. Data were tested for normality using the Sapiro-Wilk test, and one-way ANOVA was used to test for significance. The p-values are displayed.

Article Snippet: Thereafter, cells were stained overnight at 4°C with a primary antibody for SYT1 (SYSY Antibodies, 105011, 1:500), LAMP1 (Sigma, L1418,1:140), VAMP3 (Novus Biological, NB300-510, 1:100) in CLSM with 0.1% saponin (Sigma, 47036-50G) at 4°C.

Techniques: Derivative Assay, Transfection, Labeling, Expressing, Incubation, Membrane, Immunolabeling, Marker, Fluorescence

Journal: Materials Today Bio

Article Title: Engineering exosomes from fibroblast growth factor 1 pre-conditioned adipose-derived stem cells promote ischemic skin flaps survival by activating autophagy

doi: 10.1016/j.mtbio.2024.101314

Figure Lengend Snippet: The expression of FGF1 and the identification and comparison of EXO and FEXO. (A) The volcano plot of different mRNAs of human skin flaps. (B) The transcriptome sequencing analysis of human skin flaps. (C) Representative signaling pathways of genes targeted by significantly differential mRNAs according to GO analysis. (D) The transcriptome sequencing analysis of FGFs family in human skin flaps, and the upregulated FGFs in necrotic area were labeled by red rectangle. (E) qPCR result of FGF1, FGF2, FGF9, FGF12, FGF14, FGF16, FGF18, FGF21, FGFR1, FGFR2, FGFR4,FGF7 and FGF11 in necrotic and survived area of mice skin flaps (n = 5). (F) The western blot images of FGF1 in human skin flaps and mice skin flaps. (G)The transmission electron microscopy image of EXO and FEXO. (H) Uptake of Dil-labeled EXO and FEXO into OGD endothelial cells. (I) The particle distribution of EXO and FEXO by NTA analysis. (J) Particle sizes of EXO and FEXO were analyzed (n = 3). (K) Representative signaling pathways of genes targeted by significantly differential microRNAs according to GO analysis of FEXO versus EXO. (L) Western blot analysis of biomarkers of EXO and FEXO. Extracellular vesicle positive marker CD81 and CD9 and negative control calnexin. The gels have been run under the same experimental conditions, and cropped blots were used here. (M) The density value of the proteins previously described in each group (n = 3). (N) The western blotting of VAMP3 in ADSCs, F-ADSCs, F-ADSCs si-NC , F-ADSCs si-VAMP3 . The gels have been run under the same experimental conditions, and cropped blots were used here. (O) The density value of VAMP3 in each group (n = 3). (P) The number of particles produced by each ADSC was analyzed by NTA in ADSC and F-ADSC groups (n = 5). (Q) The viability of OGD endothelial cells by CCK8 analysis (n = 5). (R) The Elisa results of FGF1 in patients serum (n = 3). ∗ stands for P < 0.05, ∗∗ stands for P < 0.01, ∗∗∗ stands for P < 0.001, ns stands for not significant.

Article Snippet: The primary antibodies are following: VEGF (1:1000, Abcam, ab214424), LC3B (1:1000, Huabio, ET1701-65), p62 (1:1000, Proteintech, 66184-1-Ig), NLRP3 (1:1000, Proteintech, 27458-1-AP), GSDMD-N (1:1000, Affinity, DF13758), Cleaved Caspase1 (1:1000, Affinity, AF4005), GPR137 (1:1000, Proteintech, 11929-1-AP), mTOR (1:1000, Huabio, ET1608-5), P-mTOR (1:1000, Huabio, HA721632), PI3K (1:1000, Huabio, HA721673), P-PI3K (1:1000, Abclonal, AP0427), AKT (1:1000, Huabio, ET1609-51), P-AKT (1; 1000, Huabio, ET1607-73) and VAMP3 (1:1000, Abclonal, A8812), FGF1 (1:1000, Zenbio, R24301), GAPDH (1:50000, proteintech, 60004-1-IG).

Techniques: Expressing, Comparison, Sequencing, Protein-Protein interactions, Labeling, Western Blot, Transmission Assay, Electron Microscopy, Marker, Negative Control, Produced, Enzyme-linked Immunosorbent Assay