Journal: Viruses

Article Title: Cargo and Biological Properties of Extracellular Vesicles Released from Human Adenovirus Type 4-Infected Lung Epithelial Cells

doi: 10.3390/v17101300

Figure Lengend Snippet: Characterization of the EV populations isolated from the mock- and AdV-infected A549 cells. ( A ) Phase contrast images of the A549 cells showing the magnitude of cytopathic effect at 24 h post-infection. Cells were infected at MOI = 10. Scale bars = 100 µm. ( B ) The representative Western blot analysis showing enrichment of the EV markers CD63, CD9, CD81, Alix in EVs-Ctr and EVs-AdV, compared to their cells of origin (cell lysates). The ER marker GRP94 was used to assess the presence of non-EV contaminants. Images are representative of three different experiments. ( C ) The TEM images of EVs isolated by magnetic immunocapture form the supernatants of mock-infected cells (Ctr-EVs) or HAdV-E4-infected (AdV-EVs) A549 cells. The magnetic beads are recognizable by their opaque appearance and uniform size of 50 nm (black arrows). Scale bars = 200 nm. ( D ) The TEM image of the purified HAdV-E4 particles (white arrows). Scale bar = 200 nm. ( E ) The fluorescent nanoparticle tracking analysis of Ctr-EVs (left panel), AdV-EVs (middle panel), and magnetic beads alone (right panel).

Article Snippet: EVs were isolated from the concentrated medium with EV isolation kits Pan (human), or CD81 human (Cat # 130-111-572 and 130-110-914, Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions.

Techniques: Isolation, Infection, Western Blot, Marker, Magnetic Beads, Purification

Journal: Cancer Research

Article Title: Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

doi: 10.1158/0008-5472.can-11-2520

Figure Lengend Snippet: Figure 3. Phosphorylation and dephosphorylation of Thr443. A, in vitro kinase assay was carried out with the CS-PKA on GST fusion proteins containing the IC domain of CA IX in the wt version and in the mutated version with Thr443 replaced by Ala (T443 ! A). The upper part of the figure represents a CBB-stained gel (loading control), the lower part represents autoradiograph of the proteins phosphorylated in the presence of [g-32P]ATP. B, in vitro phosphatase assay was carried out with heterodimeric and heterotrimeric forms of PP2A on PKA-phosphorylated GST-tagged wt-IC fragment of CA IX. The upper part of the figure represents a gel with CBB-stained substrate proteins (loading control), the lower part represents autoradiograph. The data show that Thr443 is a substrate residue for PKA and PP2A. The experiments (in A and B) were repeated twice and representative results are shown. C, CA IX was immunoprecipitated by the M75 MAb from cell extracts, in vitro phosphorylated by CS-PKA, subjected to PAGE, blotted and incubated with the P-T443 CA IX antibody. In vitro phosphorylated GST-tagged wt-IC domain of CA IX protein (GST-IC) was used as a positive control. D, all 3 blots contain PAGE-resolved extracts from HEK293T/17 cells transfected with different combinations of the following plasmids: pcDNA3.1 (p1), pSG5C (p2), pSG5C-MN/CA9 (pCA9), pcDNA3.1- CSPKA (pCa), pcDNA3.1dnPKA (pdn). The plasmids were cotransfected in a ratio of 10:1. The blots were incubated with antibodies as indicated on the left side. The data show that phosphorylation of CA IX, activation of PKA and phosphorylation of PKA substrates occur only in the presence of CS-PKA. The experiment was repeated 4 times.

Article Snippet: HEK293T/17 cells were cotransfectedwith various combinations of the following plasmids: pCalpha-EV-PKA encoding CSPKA (pCa, catalytic subunit C alpha, 15,310 Addgene plasmid; ref. 17), pcDNA3.1þdnPKA encoding DN-PKA (pdn), pSG5CMN/CA9 encoding CA IX (pCA9) and respective empty plasmidsbyNeoFectin in the ratio10:1, 4mgofplasmidDNA in total.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, In Vitro, Kinase Assay, Staining, Control, Autoradiography, Phosphatase Assay, Residue, Immunoprecipitation, Incubation, Positive Control, Transfection, Activation Assay

Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: Study design and characterization of plasma-derived extracellular vesicles. A Schematic illustration of the study design and workflow. Fifty-nine patients with breast cancer were included in the study. Blood samples were collected at two time points: before NST initiation (PRE) and after completion of NST, prior to surgery (POST). Pathological response was evaluated post-surgery, and patients were classified as either achieving pathological complete response (pCR) or having residual disease (non-pCR). Patients were stratified into breast cancer subtypes (Luminal B-like (LUMB), HER2-positive, and triple-negative breast cancer (TNBC)), and clinical data were recorded. EVs were isolated from 1 mL of plasma using size exclusion chromatography (SEC) followed by ultrafiltration. Downstream EV characterization and analysis included transmission electron microscopy (TEM), western blotting, nanoparticle tracking analysis (NTA), Qubit protein quantification, and MACSPlex surface marker profiling. B TEM image showing vesicle-like structures with typical morphology and diameters below 200 nm. Smaller, bright particles were also observed, likely corresponding to co-isolated lipoproteins or other non-vesicular components. C Western blot analysis of EV preparations from three patients (PRE and POST). The EV markers CD9 and Flotillin were detected in the EV samples, while the cellular marker Calnexin was absent. The plasma protein ApoA1 was present in all EV preparations, indicating co-isolation of plasma components. Cell lysate was used as a control for CD9, Flotillin and Calnexin, while plasma was used for ApoA. Stain-free SDS PAGE gels used to verify equal loading for Western blot analysis are shown in Additional file

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Clinical Proteomics, Derivative Assay, Isolation, Size-exclusion Chromatography, Transmission Assay, Electron Microscopy, Western Blot, Marker, Control, Staining, SDS Page

Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: Pre-treatment EV surface marker profiles across breast cancer subtypes and treatment response groups. Surface marker expression in plasma-derived EVs collected before neoadjuvant systemic treatment (PRE), analyzed using the MACSPlex assay. Values represent CD9/CD63/CD81-normalized median fluorescence intensity (MFI, APC channel) for markers retained after threshold filtering (MFI > 0.5 in ≥ 50% of samples). A Marker expression grouped by breast cancer subtype: Luminal B (LUMB), HER2-positive (HER2 +), and triple-negative breast cancer (TNBC). B Marker expression grouped by treatment response: pathological complete response (pCR) vs. non-pCR. Note that all EV samples were collected before treatment, while response was evaluated after completion of NST. Bars represent mean values with standard deviation (SD). Statistical comparisons were performed using one-way ANOVA or unpaired t-tests, with multiple comparisons corrected using the Holm method. A p -value < 0.05 was considered statistically significant and is indicated by *; non-significant results are labeled as “ns”. Individual marker data are shown in Additional files (subtypes) and (treatment response)

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Marker, Expressing, Clinical Proteomics, Derivative Assay, Fluorescence, Standard Deviation, Labeling

Journal: Breast Cancer Research : BCR

Article Title: Plasma extracellular vesicles reflect response and prognosis in patients with breast cancer undergoing neoadjuvant treatment

doi: 10.1186/s13058-025-02209-0

Figure Lengend Snippet: EV surface marker expression changes in response to neoadjuvant systemic treatment. Surface marker expression in plasma-derived EVs before and after neoadjuvant systemic treatment (NST), assessed using MACSPlex analysis. For each patient and marker, data are presented as the POST/PRE ratio of CD9/CD63/CD81-normalized APC signal, representing the individual fold change. A All patients combined. B Stratified by treatment response: pathological complete response (pCR) vs. non-pCR. Graphs show show box plots with median, interquartile range, and full range. All data are plotted on a log scale; values > 1 indicate increased expression post-treatment, and values < 1 indicate decreased expression. Statistical comparisons were performed using paired t-tests with Holm correction. Colored asterisks indicate significant within-group changes relative to baseline (PRE), red asterisks indicate significant upregulation post-treatment, and blue asterisks indicate significant downregulation post-treatment. *Significance level: * p < 0.05

Article Snippet: Surface protein profiling of EVs was performed using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer’s overnight protocol for the MACSPlex Filter Plate with minor modifications.

Techniques: Marker, Expressing, Clinical Proteomics, Derivative Assay