Journal: Stem cell research & therapy

Article Title: Exosomes derived from hypoxic mesenchymal stem cells restore ovarian function by enhancing angiogenesis.

doi: 10.1186/s13287-024-04111-6

Figure Lengend Snippet: Fig. 2 Characterization and internalization of norm-Exos and hypo-Exos. A. Morphology of norm-Exos and hypo-Exos observed using TEM. B. Particle size distributions were determined by NTA. C. Comparison of the mean diameters of norm-Exos and hypo-Exos. D. The exosomal surface markers (TSG101, CD9, CD63, and CD81) in norm-Exos and hypo-Exos were assessed by western blotting. E. Semiquantitative analysis of the protein levels of TSG101, CD9, CD63, and CD81. F. Exosomal protein concentrations in norm-Exos and hypo-Exos were analysed using the BCA assay. G. Uptake of PKH67-labelled norm-Exos and hypo-Exos into ROMECs at 24 h. H. Statistical evaluation of fluorescence intensities in the norm-Exo and hypo-Exo groups. n = 3 per group, *P < 0.05 for all figures

Article Snippet: The resulting protein bands were visualized by the enhanced chemiluminescence detection system (Millipore, USA) The main antibodies used in this study are as follows: Calnexin (1:2000, Affinity, China), TSG101 (1:2000, Proteintech, China), CD9 (1:1000, Proteintech, China), CD63 (1:1000, Proteintech, China), CD81 (1:1000, Proteintech, China), GAPDH (1:5000, Proteintech, China), PTEN (1:2000, Affinity, China), PI3K (1:2000, Cell Signaling Technology, USA), p-PI3K (1:2000, Cell Signaling Technology, USA), AKT (1:2000, Cell Signaling Technology, USA), p-AKT (1:2000, Cell Signaling Technology, USA), mTOR (1:2000, Cell Signaling Technology, USA), and p-mTOR (1:2000, Cell Signaling Technology, USA).

Techniques: Comparison, Western Blot, BIA-KA, Fluorescence

Journal: Frontiers in Immunology

Article Title: R3HDM4 influences kidney renal clear cell carcinoma progression, immune modulation, and potential links to the IGSF8 immune checkpoint

doi: 10.3389/fimmu.2025.1722358

Figure Lengend Snippet: R3HDM4 silencing modulates expression of EMT, invasion, and immune-related markers in 786-O cells. (A) Representative Western blot of E-cadherin, MMP-2, MMP-9, Vimentin, IGSF8 and GAPDH in 786-O (control), 786-O+si-NC (negative control siRNA), and 786-O+si-R3HDM4-4 (R3HDM4-targeting siRNA) groups. (B) Quantification of relative E-cadherin expression across 786-O, si-NC, and si-R3HDM4–4 groups. (C) Quantification of relative MMP-2 expression across 786-O, si-NC, and si-R3HDM4–4 groups. (D) Quantification of relative MMP-9 expression across 786-O, si-NC, and si-R3HDM4–4 groups. (E) Quantification of relative Vimentin expression across 786-O, si-NC, and si-R3HDM4–4 groups. (F) Quantification of relative IGSF8 expression across 786-O, si-NC, and si-R3HDM4–4 groups. P - values were determined by one-way ANOVA; ** P < 0.001 indicates statistical significance.* P < 0.05, ** P < 0.01, *** P < 0.001. CTRL, control untreated; si-NC, negative control siRNA; si-R3HDM4, R3HDM4-targeting siRNA; E-cadherin, epithelial cadherin; MMP-2/9, matrix metalloproteinase-2/9; IGSF8, Immunoglobulin Superfamily Member 8 expression.

Article Snippet: The primary antibodies used were: R3HDM4 (29 kDa, 1:1000, Invitrogen, USA), GAPDH (36 kDa, 1:10,000, Proteintech, China), E-cadherin (125 kDa, 1:40,000, Proteintech, China), Vimentin (55 kDa, 1:40,000, Proteintech, China), MMP2 (63 kDa, 1:1000, BIOSS, China), MMP9 (78 kDa, 1:1000, Affinity, USA), and IGSF8 (70 kDa, 1:2000, Proteintech, China).

Techniques: Expressing, Western Blot, Control, Negative Control

Journal: Cells

Article Title: Calmodulin as a Key Regulator of Exosomal Signal Peptides

doi: 10.3390/cells12010158

Figure Lengend Snippet: Interaction of CaM with HSP70 and CD81 in T-REx cells ( A ) The peptide solution extracted from immunoprecipitants with the CaM antibody from T-REx cells was analyzed by MALDI-TOF-MS. ( B ) The peak intensity at m/z 861 was measured by MALDI-TOF-MS. ** p < 0.01 vs. Mock cells in the absence of W13 and EGTA. C; control, Mock; T-REx Mock, AspALP; T-REx AspALP. ( C ) Western blot analysis of immunoprecipitants with the CaM antibody from T-REx cells. Representative images from three independent experiments.

Article Snippet: Primary antibodies were directed against CD81 (Novus Biologicals, Centennial, CO) and CD63 (Novus Biologicals) under non-reducing conditions and against CaM (Abcam, Cambridge, UK), Alix (Cell Signaling Technology), HSP70 (Enzo Life Sciences, Farmingdale, NY, USA), and GAPDH (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan).

Techniques: Control, Western Blot

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Cellular response and extracellular vesicles characterization of human macrophages exposed to fine atmospheric particulate matter.

doi: 10.1016/j.envpol.2019.07.101

Figure Lengend Snippet: Fig. 3. Characterization of extracellular vesicles derived from THP-1 exposed to PM2.5-0.3. EVs were obtained from THP-1 exposed 6 h, 24 h or 48 h to 0 (NE), 12.5 or 50 mg/mL of PM2.5-0.3. A) Graphs representing the amounts and the mean of size of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p < 0.01); B) Transmission electronic microscopy images of EVs (bar ¼ 100 nm); C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition.

Article Snippet: After blocking, membranes were incubated with anti-CD63 or anti-CD81 primary antibodies (Novus Biologicals, Littleton, CO, USA) at 4 C overnight, washed with PBSTween (0.05% Tween 20), and incubated with a Goat anti-Mouse IgG (HþL) secondary antibody, Horseradish Peroxidase (HRP) conjugated (Life Technologies, ThermoFisher Scientific, Illkirch, France) for 60 min. After washing with PBS-Tween, antigeneantibody reactions were detected with a Chemidoc (Biorad, Marnes la Coquette, France) using the ECL Plus Detection kit (Pierce, ThermoFisher Scientific, Illkirch, France).

Techniques: Derivative Assay, Transmission Assay, Microscopy, Western Blot

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Cellular response and extracellular vesicles characterization of human macrophages exposed to fine atmospheric particulate matter.

doi: 10.1016/j.envpol.2019.07.101

Figure Lengend Snippet: Fig. 6. Characterization of extracellular vesicles derived from primary macrophages exposed to PM2.5-0.3. EVs were obtained from pooled supernatants of primary macrophages isolated from 6 donors and exposed 48 h to 0 (NE) or 12.5 mg/mL of PM2.5-0.3. A) Graph representing the amounts of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p < 0.01); B) Graphical representation of size distribution of EVs produced by non exposed (NE) and exposed (12.5 mg/mL) primary macrophages; C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition; D) Transmission electronic microscopy images of EVs (bar ¼ 100 nm except when indicated).

Article Snippet: After blocking, membranes were incubated with anti-CD63 or anti-CD81 primary antibodies (Novus Biologicals, Littleton, CO, USA) at 4 C overnight, washed with PBSTween (0.05% Tween 20), and incubated with a Goat anti-Mouse IgG (HþL) secondary antibody, Horseradish Peroxidase (HRP) conjugated (Life Technologies, ThermoFisher Scientific, Illkirch, France) for 60 min. After washing with PBS-Tween, antigeneantibody reactions were detected with a Chemidoc (Biorad, Marnes la Coquette, France) using the ECL Plus Detection kit (Pierce, ThermoFisher Scientific, Illkirch, France).

Techniques: Derivative Assay, Isolation, Produced, Western Blot, Transmission Assay, Microscopy

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: Characterization of EV. Particle numbers (A) and relative protein concentrations (B) in the fractions eluted following qEV size-exclusion chromatography. Data are mean ± SEM. (C) Tetraspanin (CD9, CD63, and CD81) content of purified EVs was analyzed in the indicated elution fractions by immunoblotting. (D) Size distribution of pooled fractions 7 to 10 from HDFs (left panel) and L3.6 cells (right panel). (E) Electron microscopy image of EV from HDFs (left panel) and L3.6 cells (right panel).

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Size-exclusion Chromatography, Purification, Western Blot, Electron Microscopy

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: polyP on EV derived from cancer cells mediates ligand binding. (A) The amount of polyP associated with cancer cell–derived EV was estimated by comparison with a standard curve (supplemental Figure 2A). (B) Detection of polyP on L3.6-derived EVs following incubation with buffer (HBS), DNase I and RNase A (D/R), or CIP. polyP of different chain lengths was used to estimate the size of EV-associated polyP. L3.6-derived EVs were preincubated with buffer or E coli PPX, and the binding of PPX-Δ12–AF488 (C), anti-CD81–AF488 (D), and or FXII-AF488 (E) was measured. The number of total EV was determined in scatter mode, and the number of ligand-binding EV was determined in fluorescence mode using a 488-nm excitation laser. The differences in fluorescent particle numbers were compared by estimating the area under each curve (AUC) using GraphPad Prism 8. Bars represent means ± SEM. *** P < .001, 1-way ANOVA with multiple comparisons. L, long; M, medium; S, short.

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Derivative Assay, Ligand Binding Assay, Comparison, Incubation, Binding Assay, Fluorescence