Journal: STAR Protocols

Article Title: Protocol for large-scale, high-yield, high-purity extracellular vesicle purification from human plasma

doi: 10.1016/j.xpro.2026.104428

Figure Lengend Snippet: Fusion assay demonstrating the uptake of EVs from different fractions by cells EV populations were stained with DiI dye, then incubated with hTERT-HUVEC cells for 16 h. Cells were then fixed and DNA was stained with DAPI. Images taken at 20X and 100X magnification. Scale bar=10 μm

Article Snippet: hTERT-HUVEC (Human Telomerase Reverse Transcriptase (hTERT), Human umbilical vein endothelial cells (HUVEC)) , ATCC , CRL-4053 TM.

Techniques: Single Vesicle Fusion Assay, Staining, Incubation

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) , ATCC , Primary cells, pooled donors.

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Migration, Activity Assay, Software

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Effects of HSPB1 on signaling pathways and TGF-β secretion in HUVECs under hypoxic conditions (A and B) HUVECs were transfected with adenoviral vectors for HSPB1 overexpression (OE) or knockdown (KD) and cultured for 48 h before RNA extraction. Gene expression analysis was performed using RNA sequencing. Gene set enrichment analysis (GSEA) assessed the regulatory roles of HSPB1 in processes such as heart development, angiogenesis, and cell proliferation (A). Further analysis using Hallmark gene sets explored HSPB1 signaling pathway activation (B). (C–G) Following transfection, HUVECs were cultured for 24 h and subjected to hypoxic conditions (3% O 2 ) for 48 h. Western blot analysis of the indicated proteins was performed. (D) pSmad2/3/Smad2/3 ratio, (E) quantification of CD31 protein expression, (F) quantification of E-cadherin expression, (G) quantification of α-SMA expression, and (H) quantification of N-cadherin expression were measured relative to β-actin. (I) TGF-β levels were measured by ELISA in cell supernatants. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) , ATCC , Primary cells, pooled donors.

Techniques: Protein-Protein interactions, Transfection, Over Expression, Knockdown, Cell Culture, RNA Extraction, Gene Expression, RNA Sequencing, Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Expression of ACKR1 mRNA in mouse lung endothelial cells (L-ECs), E0771 and E0771.LMB (LMB) cells. Significance determined by one-way ANOVA. (B) Representative image of Day 28 LMB tumor in a wildtype mouse, showing adjacent mammary stroma. Dotted line indicates border between tumor (right side of image) and stroma (left side of image). Rectangle indicates inset area adjacent. CD31 = magenta, ACKR1 = green. ACKR1 is expressed in some stromal vessels (red arrowheads). (C-E) Tumor growth curve and final weights of LMB and PY8119 tumors implanted into the 4 th mammary fat pad of WT and Ackr1 Null mice. Significance determined by multiple unpaired t-test (C, E) or unpaired t-test (D). (F) Representative low magnification overviews of lung sections of wildtype and Ackr1 Null mice implanted with LMB tumors, Day 28 endpoint. Red = RFP pos tumor cells. Same mice as details shown in .

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Expressing

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative images of spontaneous lung metastases from orthotopic E0771.LMB (LMB) mammary tumors implanted into WT and Ackr1 Null mice harvested at humane endpoint (2 cm tumor diameter), Day 28 after tumor injection. Tumor cells are detected with mCherry (red) with individual tumor cells (white arrowheads) indicated in higher magnification inset. (B) Quantification of lung metastases in WT (n = 6) and Ackr1 Null (n = 7) mice. Error bars indicate s.e.m. in all figures. Significance determined by unpaired t-test. Each data point represents a single mouse, in which a minimum of three sections at different depths within the lung have been analyzed and averaged. (C) Representative lung sections from tumor-free (TF) mice and mice bearing LMB and AT3 orthotopic mammary tumors harvested at humane endpoints (Day 28 and Day 40, respectively). Immunofluorescent (IF) staining of endothelial cells with CD31 (magenta), ACKR1 (green) and DAPI (blue). ACKR1-expressing vessels indicated with white arrowheads. (D) Quantification of ACKR1-positive vessels per mm 2 in TF, LMB-bearing, and AT3-bearing mouse lungs. Significance determined by one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Injection, Staining, Expressing

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative IF images of lungs from C57BL6/J wildtype mice at 3, 7, 13, and 28 days after orthotopic injection with LMB cells or tumor-free (TF) PBS mock injection. CD31 = magenta, ACKR1 = green, DAPI = blue in all panels. ACKR1-positive endothelial cells indicated by white arrowheads. Fainter ACKR1 staining can also be seen in red blood cells. (B) Representative IF images of lungs from non-transgenic or MMTV-PyMT transgenic C57BL6/J mice at 8-10 weeks (hyperplasia stage) and 18-22 weeks (late stage carcinoma). (C) Number of disseminated LMB tumor cells per mm 2 in lungs at indicated timepoints after tumor implantation. One-way ANOVA with Sidak’s multiple comparisons test. (D) Average number of ACKR1-positive vessels per lung section at indicated timepoints after tumor implantation. Unpaired t-test. (E) Quantification of ACKR1-positive vessels per mm 2 in lungs of non-transgenic (18 week old), MMTV-PyMT transgenic at hyperplasia stage (8-10 week old, HYP) and MMTV-PyMT transgenic at late stage carcinoma (18-22 week old, LSC) mice. One-way ANOVA with Sidak’s multiple comparisons test. For all panels, each data point represents a single mouse, in which a minimum of three sections at different depths within the lung have been analyzed and averaged.

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Injection, Staining, Transgenic Assay, Tumor Implantation

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative image of ACKR1-positive lung vessel from LMB-bearing mouse 28 days after implantation, showing colocalization of ACKR1 with venous endothelial marker EphB4. CD31 = magenta, EphB4 = yellow, ACKR1 = green. All ACKR1 pos vessels also expressed EphB4. 22.7±1.4% of EphB4 pos venules expressed ACKR1. Quantitation shown in . (B) Representative annotated IF image for QuPath-based proximity analysis of tumor cell distance to nearest venule (EphB4 = yellow, Pan-CK = tumor cell marker pancytokeratin in red, ACKR1 = green, and DAPI = blue). For QuPath annotations, yellow line = vessel border, red circle = disseminated tumor cell (DTC), red line = shortest distance between DTC and nearest venule border. (C) Quantification of the number of DTCs within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 116 ACKR1 pos venules, n = 100 ACKR1 neg venules, across 4 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in . (D) Percentage of ACKR1-positive and ACKR1-negative venules with at least one DTC within 50 µm of the venule border. Chi-squared test. (E) Representative IF images for QuPath proximity analysis of neutrophil distance to nearest venule, as described in panel B except Ly6G = neutrophil marker (cyan) and red lines indicate shortest distance between neutrophil and nearest venule border. (F) Quantification of the number of neutrophils within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 16 ACKR1 pos venules, n = 16 ACKR1 neg venules, across 3 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in .

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Marker, Quantitation Assay

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Schematic of the Ackr1 floxed allele and its excision in endothelial cells following tamoxifen-induced Cdh5CreERT2 recombination to generate the Ackr1 ECKO mouse model. Consensus GATA/TAL binding sequences (black triangles) were not disrupted when inserting the loxP sites (red triangles). (B) Representative IF images (n=2 mice per genotype) of peripheral lymph nodes, which constitutively express ACKR1 in endothelial cells, confirming the loss of ACKR1 protein in Ackr1 ECKO mice (CD31 = magenta, ACKR1 = green). (C) Flow cytometry analysis of peripheral blood confirms retention of ACKR1 expression in red blood cells of Ackr1 ECKO mice. TER-119 = red blood cell marker, anti-ACKR1 = blue peaks, isotype control = red peaks. Control = Cdh5CreERT2;Ackr1 WT , Null = Ackr1 Null (negative control), Flox = Ackr1 flox/flox (no cre control), ECKO = Cdh5CreERT2;Ackr1 flox/flox (experimental). (D) Representative images of Day 28 LMB mammary tumor-bearing Control and Ackr1 ECKO mouse lungs confirming loss of endothelial ACKR1 in Ackr1 ECKO mouse lungs after induction by primary mammary tumors (CD31 = magenta, ACKR1 = green). (E) Quantification of loss of ACKR1-positive vessels in Day 28 LMB tumor-bearing Control (n=3) and Ackr1 ECKO (n=4) mouse lungs. Unpaired t-test.

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Binding Assay, Flow Cytometry, Expressing, Marker, Control, Negative Control

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Schematic of extravasation assessment protocol. 300 µl TFM or TCM-LMB was administered daily via IP injection, then LMB cells were administered on day 9 via retroorbital injection. 24 hours after tumor cell injection, anti-MHCI (H-2kB) antibody was retroorbitally injected to label intravascular tumor cells and allowed to circulate for 3 minutes before sacrifice. (B) Flow cytometry quantifying total tumor (RFP pos ) cells in the lung. Each dot represents one mouse. One-way ANOVA with Tukey’s multiple comparisons. The same mice are represented in panels B-F. (C) Percentage of extravasated (RFP pos , MHC1 neg ) tumor cells in the lung. Unpaired t-tests. (D) Representative gating strategy for neutrophils (CD45+/Gr-1+). (E) Quantitation of neutrophil infiltrate, expressed as percentage of total lung cells. Unpaired t-tests. (F) Contribution of neutrophils to the total immune cell infiltrate (CD45+). Unpaired t-tests. (G) Representative immunohistochemistry images of lung neutrophils (Ly6G, brown). (H) Neutrophils per mm 2 in lung tissue sections. Unpaired t-test. (I) Schematic illustrating the design of neutrophil transmigration assay. HUVEC were grown to confluence onto a collagen gel and cultured for 24 hours in TFM or MDA-TCM. HUVEC were either left unstimulated or activated with TNFα for 4 hours. Freshly harvested neutrophils (PMNs) were seeded on top and allowed to migrate for 6 minutes. Cultures were rinsed with 1mM EDTA to remove loosely adherent cells, then fixed and stained. In Z-stack images, neutrophils that were in focus in the same plane as endothelial cells (black arrowheads) were scored as surface adherent (red arrows) and those in focus in deeper planes were scored as transmigrated (brown arrows). (J) Quantitation of total adhered (surface adherent + transmigrated) and transmigrated neutrophils in TFM or TCM-MDA treated monolayers without TNFα treatment. Each data point represents the mean count of at least 12 high power fields of view (FOV). Unpaired t-test.

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Injection, Flow Cytometry, Quantitation Assay, Immunohistochemistry, Neutrophil Transmigration Assay, Cell Culture, Staining

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Confirmation of MHCI labeling of intravascular tumor cells. Flow analysis of blood from animals in , showed that 99% of intravascular tumor cells were positive for anti-MHCI. (B) To examine antibody leakage to the extravascular space in the lung, we intravenously injected an antibody to collagen type IV (Col4), which does not have a target when confined to the endothelial lumen but readily binds the abluminal endothelial basement membrane upon leakage . Mice were intratracheally injected with PBS (control) or 300 ng TNFα to induce lung inflammation. After 24 hours, anti-Col4 was administered via retroorbital injection three minutes prior to euthanasia. No Col4 leakage was observed in baseline or inflamed lungs (top left and center). Fenestrated liver endothelium acted as a positive control for Col4 leakage (bottom left and center). IF staining on sections confirmed Col4 immunoreactivity in both tissues (top and bottom right). (C) Absolute neutrophil counts (ANC) from automated complete blood counts (CBCs) of mice treated with TFM or TCM-LMB for 9 days, then injected retroorbitally with LMB tumor cells 24 hours prior to sacrifice (see , panels A-F). Circulating neutrophil levels were not reduced in ECKO animals. (D) Representative images of neutrophil transendothelial migration assays with TFM- or TCM-MDA-treated HUVEC. Red arrowheads indicate neutrophils, which may be in focus or out of focus in each focal plane. (E) Quantitation of total adhered (surface adherent + transmigrated) and transmigrated neutrophils in monolayers treated with TFM or TCM-MDA for 24 hours and then activated with TNFα 25 ng/mL for 4 hours prior to seeding with neutrophils. Each data point represents the mean count of at least 12 high power fields of view (FOV). Unpaired t-test.

Article Snippet: Human Umbilical Vein Endothelial Cells from pooled donors (HUVECs, Promocell, C-12203) were cultured in Endothelial Growth Medium 2 (EGM, Promocell, C-22011) on Type I Collagen-coated plates at 37°C with 5% CO 2 .

Techniques: Labeling, Injection, Membrane, Control, Positive Control, Staining, Migration, Quantitation Assay