Journal: bioRxiv

Article Title: Systemic ligand-mimicking bioparticles cross the blood-brain barrier and reduce growth of intracranial triple-negative breast cancer using the human epidermal growth factor receptor 3 (HER3) to mediate both routes

doi: 10.1101/2021.06.07.446634

Figure Lengend Snippet: A, Bioinformatics analysis comparing HER3 gene expression of all invasive breast cancers including TNBC (TCGA-1097 database; N=199 samples) against normal breast tissue (Roth database; N=17 samples) using the R2 Genomics Analysis and Visualization Platform ( http://r2.amc.nl ). B, Gene expression heatmap of 198 TNBC tumors (Brown database) queried for HER3 (ERBB3), HER2 (ERBB2), estrogen receptor (ESR1) and progesterone receptor (PR) expression. Samples are ranked by HER3 expression levels. Percent of cases showing above average HER3 (defined by average HER3 expression in normal breast) are demarcated above the heatmap. C, Flow cytometry measurement of cell surface HER3 on human [BT549, MDA-231(+), MDA-231(-)] and mouse (4T1) TNBC lines. D, Immunocytofluorescence of HER3+ TNBC cells [MDA-231(+)] in comparison to respective cells with no/low HER3 expression [MDA-231(-)]. Scale bar, ~8 μm. E, Immunocytofluorescence of patient-derived tumor cells. Scale bar, ~8 μm. F, Graphical representation of the HPK linear sequence from amino [N] to carboxy [C] terminus (left to right), highlighting: HER3-binding motif (Her) appended to a flexible linker sequence comprised of neutral residues (Gly-Gly-Ser) 2 followed by the penton base sequence (PB) and decalysine (K10). G, Ribbon models of monomeric and pentameric HPK generated by molecular dynamics (MD) simulation, with each functional region depicted by a designated color. Inset, transmission electron micrograph (TEM) of HPK capsomeres. H, Cell surface ELISA (graph) showing HPK binding to human (BT549) and mouse (4T1) TNBC cells -/+ competing HER3 peptide. ****, p<0.0001. Data show individual measurements from quadruplicate samples and corresponding means. Inset, graphical alignment of mouse and human HER3 ligand binding domains (amino acid residues 8-239) showing identical, similar, and mismatched residues. I, Video stills of MD-simulated HPK pentamer (with each monomer distinguished by a different colored ribbon structure) in a neutral solution with titrating H+ added over time. Full video of capsomere dynamics is shown in Supplemental Movie 1 . J, Immunocytofluorescence of 4T1 cells at 30 min after uptake of HPK capsomeres -/+ bafilomycin-A1. Scale bar, ~8 μm. Graph summarizes contrast between vesicular (v) and extravesicular (e) regions by measuring integrated densities (Int D) of each and applying the formula shown by the y-axis label. K , Intracellular trafficking of HPK capsomeres in HER3+ MDA-MB-435 cells in relation to early endosomes (EEA1) and late (RAB7) endo-lysosomes. Scale bar, ~5 μm. Graph summarizes the intracellular distribution of HPK per cell across all time points as a % of internalized HPK. L, Immunoblots of subcellular fractions isolated from HER3+ MDA-MB-435 human tumor cells harvested and processed at the indicated time points during uptake of HPK or HΔPK. Relative levels of uptake are quantified by normalizing band densitometries with those of respective fraction controls. Where indicated, fold change is reflected by the difference in normalized band densitometry at subsequent time points compared to that at 0 min.

Article Snippet: Slides were blocked in 1% BSA and then stained with indicated antibodies against Claudin-5 (Invitrogen 35-2500 1:50), Ad5 (Abcam ab6982 1:100), HIS-tag (Qiagen 34610 1:100) and HER3 (R&D Systems AF4518 1:400).

Techniques: Gene Expression, Expressing, Flow Cytometry, Comparison, Derivative Assay, Sequencing, Binding Assay, Ubiquitin Proteomics, Generated, Functional Assay, Transmission Assay, Enzyme-linked Immunosorbent Assay, Ligand Binding Assay, Western Blot, Isolation

Journal: bioRxiv

Article Title: Systemic ligand-mimicking bioparticles cross the blood-brain barrier and reduce growth of intracranial triple-negative breast cancer using the human epidermal growth factor receptor 3 (HER3) to mediate both routes

doi: 10.1101/2021.06.07.446634

Figure Lengend Snippet: A, Tissue distribution of near infrared (NIR)-labeled NNCs or trastuzumab (Tz) after systemic delivery in mice bearing subcutaneous JIMT-1 breast tumors. Arrows point to tumor locations in inset. Graph summarizes average radiant efficiencies collected from tissue harvested >4h after systemic delivery of each reagent. Data represent mean±SD of each corresponding tissue from 5 mice per treatment group. Tissues shown below the x-axis were acquired from representative mice of each cohort. Lu, lung; Li, liver; Ki, kidney; H, heart; Br, brain; Sp, spleen; M, muscle; Tu, tumor. B, Brain and tumor distribution of near infrared (NIR)-labeled HPK or trastuzumab (Tz) after systemic delivery in mice bearing subcutaneous JIMT-1 breast tumors. Graph summarizes average radiant efficiencies collected from tissue (shown below x-axis) harvested 2.5 hours after systemic delivery of each reagent. Data represent mean±SD of each corresponding tissue from 5 mice per treatment group. C, Tissue content of HPK bioparticles after systemic delivery of a gallium(III)-metallated corrole payload (S2Ga) in an orthotopic syngeneic mouse model of TNBC. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Ga(III) content from each digested organ or tumor. Data represents 3 separate measurements of each organ harvested from two mice each per treatment. ****, p<0.0001. D, Quantification of tumor and brain distribution at indicated time points after systemic administration of NNCs delivering NIR-ODN in mice bearing subcutaneous JIMT1 tumors. ****, p<0.0001. Data represent mean±SD of 12 samples at each time point. Images show tumors and brains from representative mice at each time point. E, Imaging and quantification of NNC and HER3 localization in relation to brain vasculature (delineated by claudin 5) at 2.5h after systemic delivery of NNCs delivering NIR-ODN. Images show channel separated and merged micrographs capturing representative mouse brain section after immunohistofluorescent stain of indicated biomarkers. Graph shows quantification of NNC and HER3 overlap in brain vessel (V) compared to parenchyma (P) from 3 independent specimens. Data represent mean±SD. F, Assessment of HPK and NIR-ODN overlap at the vasculature of two representative brain sections from experiment in E showing adjacent z-axis planes that transect the vessel (V) and the parenchyma above the vessel (P). “Free ODN” designates the detection of NIR-ODN without overlapping HPK. Graph summarizes the overlap of HPK with NIR-ODN at each location and shows the mean±SEM from 3 independent specimens. Scale bar, ~20 μm.

Article Snippet: Slides were blocked in 1% BSA and then stained with indicated antibodies against Claudin-5 (Invitrogen 35-2500 1:50), Ad5 (Abcam ab6982 1:100), HIS-tag (Qiagen 34610 1:100) and HER3 (R&D Systems AF4518 1:400).

Techniques: Labeling, Clinical Proteomics, Mass Spectrometry, Imaging, Staining

Journal: bioRxiv

Article Title: Systemic ligand-mimicking bioparticles cross the blood-brain barrier and reduce growth of intracranial triple-negative breast cancer using the human epidermal growth factor receptor 3 (HER3) to mediate both routes

doi: 10.1101/2021.06.07.446634

Figure Lengend Snippet: A, Immunohistofluorescence of frontal cortex from non-diseased adult murine and human brains showing cross sections and longitudinal views of blood vessels within brain specimens. Specimens were obtained from female adult immunodeficient mice (6+ months) and human male frontal cortex, ages 71, 68, and 76 years old (Tissue for Research Ltd.). White squares delineate claudin positive (+) and negative (-) areas. Scale bar, ~10 μm. B, Quantification of overlap between claudin-5 and HER3 in specimens shown in A . Data points represent the means of individual regions of interest in each indicated zone. C, R2 database analysis of HER3 (N=38), glucose receptor (GLUT1; N=38) and transferrin receptor (TfR; N=38) gene expression in peripheral (non-brain) endothelial tissue, showing mean and individual data points. ****, p<0.0001. D, R2 database analysis of HER3, GLUT1, and TfR gene expression in human breast cancer brain metastases (upper graph) and human peripheral (non-brain) non-tumor tissues (lower graph, summarizing collective expression scores shown in E ). Data represent mean (X) with median and interquartile range. ***, p=0.0006. ****, p<0.0001. N=31,408 per group. E, Itemized expression scores of human peripheral (non-brain) non-tumor human tissues comparing HER3, TfR and GLUT1 genes. Red line in each category indicates the mean. Dashed vertical line delineates threshold for high expression. Each y-axis tick mark represents a separate database. Multiple databases within the same category are enumerated. Sources and N of each database are listed in the Methods . Adr, adrenal; BC, B cell; Bl, blood; Col, colon; Dev, developmental; En, endothelial; Ep, epithelial; Fa, fallopian tube; Fi, fibroblasts; Leu, leukocytes; Li, liver; Lym, lymphocytes; Mp, macrophage; Ms, mesenchymal; Mn, monocytes; Mu, muscle; Pn, pancreatic; Pc, placenta; Pt, platelets; Sk, skeletal; Spr, spermatogonia; TC, T cells; Th, thymus.

Article Snippet: Slides were blocked in 1% BSA and then stained with indicated antibodies against Claudin-5 (Invitrogen 35-2500 1:50), Ad5 (Abcam ab6982 1:100), HIS-tag (Qiagen 34610 1:100) and HER3 (R&D Systems AF4518 1:400).

Techniques: Immunohistofluorescence, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Systemic ligand-mimicking bioparticles cross the blood-brain barrier and reduce growth of intracranial triple-negative breast cancer using the human epidermal growth factor receptor 3 (HER3) to mediate both routes

doi: 10.1101/2021.06.07.446634

Figure Lengend Snippet: A, Left, schematic of BBB chip architecture showing endothelial and neuronal chambers separated by porous membrane, and micro-ducts directing flow into and out of each chamber. Right, 3D imaging of chip at 24h after introduction of NNCs into the endothelial flow chamber. Scale bar, 150 μm. B, Detection of HER3 and NNCs in BBB chips at 4h after injection of NNCs delivering NIR-ODN into endothelial flow chamber. Images show cross-sectional and 2D surface views of neuronal (N) and endothelial (E) layers. Membrane pores are visible as regularly-spaced white puncta. Scale bar, 150 μm. Graph, quantification of NNCs collected from endothelial (Endo) and neuronal (Neur) chamber effluents during 4h of NNC flow into endothelial micro-chamber. Input, NNCs measured from injection micro-chamber. Data represent mean of triplicate sample effluents quantified by sandwich ELISA. C, Relative HER3 levels on proximal and distal endothelial layers from BBB chip represented in B . Data represent averages of two regions of interest (ROIs) per zone per triplicate sample. D, Relative levels of HER3 and NNC on endothelial tube (E) and neuronal layer (N) from B . Data represent averages of two ROIs per zone per triplicate sample. E, Quantification of NNC content collected from neuronal chamber effluents during 4h of NNC flow into endothelial micro-chamber -/+ blocking of HER3. NNCs were detected by sandwich ELISA using antibody capturing HPK. Data show mean and individual measurements from triplicate BBB chip samples. F, Immunofluorescence detection of NNCs in neuronal and endothelial layers after 4h of NNC flow into the endothelial micro-chamber -/+ blocking of HER3. Top panels, NNC fluorescence channel alone. Lower panels, merged fluorescence channels. Scale bar, 150 μm. Graph, integrated signal densities of NNCs in neuronal and endothelial compartments -/+ blocking of HER3. Data show the mean and individual measurements from 4 independent fields in each region. G , Comparison of NNCs delivering NIR-ODN compared to NIR-ODN alone. Upper panels, merged fluorescence channels. Lower panels, NIR channel. Graph, Integrated signal densities comparing NIR-ODN -/+ HPK in neuronal layer, proximal endothelium and distal endothelium. Data show the means from 6 independent fields in each region and mean of these individual means.

Article Snippet: Slides were blocked in 1% BSA and then stained with indicated antibodies against Claudin-5 (Invitrogen 35-2500 1:50), Ad5 (Abcam ab6982 1:100), HIS-tag (Qiagen 34610 1:100) and HER3 (R&D Systems AF4518 1:400).

Techniques: Membrane, Imaging, Injection, Sandwich ELISA, Blocking Assay, Immunofluorescence, Fluorescence, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: TRPV2 regulates cell fate in the human granulosa-like tumor cell line KGN: implications for granulosa cell tumors and cannabidiol

doi: 10.1186/s12964-026-02729-y

Figure Lengend Snippet: TRPV2 is expressed by GCTs and KGN cells and is functional in KGN cells. A TMA samples of granulosa cell tumors derived from 63 patients were subjected to immunohistochemistry targeting TRPV2 and subdivided into four categories depending on their signal intensity/density (negative (black), weak (green), moderate (blue), strong (red). Representative images of the four categories negative (upper left), weak (upper right), moderate (lower left) and strong (lower right) are shown. Scale bar 50 μm. B Primary GCT cells derived from three individual patients (GCT#1–3) express TRPV2 mRNA, as demonstrated by RT-PCR. PCR amplicon size 100 bp. -RT (no reverse transcription) and H 2 O (water instead of RNA within the reaction) served as controls. Cropped gel image is shown and original gel images are shown in Supplementary Fig. 8. C KGN cells express TRPV2 both on mRNA (upper panel) and protein level (lower panel). PCR amplicon size 100 bp. Western Blotting revealed several bands sized between 86 kDa and ~ 70 kDa; ß-Actin served as internal loading control, band size 43 kDa. Passages used between 32 and 42 (P32-42). Cropped gel and blot images are shown and original gel images are provided in Supplementary Fig. 9. D Immunocytochemistry showed TRPV2 in KGN cells with fluorescence signals in close proximity to the nucleus (arrow heads), at the periphery (asterisks) or spotted and membrane-bound (arrows). TRPV2 - green; DAPI - blue; insert with phase contrast image. Scale bar 25 µM. E Application of cannabidiol (CBD, 10 µM; green), but not the solvent control ethanol (EtOH ctrl.; gray), elicited transient calcium (Ca 2+ ) fluxes in KGN cells, as reflected by increased fluorescence intensity measured during Ca 2+ imaging. Relative fluorescence intensity over time (normalized to starting point t 0 ) of 40 examined KGN cells (mean ± SEM; mean - black line, SEM - gray dotted line), with representative live cell images displayed as pseudo-color images (black-purple - low Ca 2+ levels; yellow-white - high Ca 2+ levels) at the indicated time points (a-d). Scale bar 50 μm

Article Snippet: Briefly, 10–15 μg protein were separated in a 10–15% SDS-PAGE and transferred to nitrocellulose membranes (AmershamTM ProtranTM, 0.45 μM; Thermo Fisher Scientific), which were incubated overnight at 4 °C with primary polyclonal rabbit IgG antibodies targeted either against TRPV2 (1:400; Atlas Antibodies), VDAC1 (Proteintech), cleaved Caspase 3 or 8 (clCasp3, #9664T, 1:1,000; clCasp8, #98134T, 1:1,000; Cell Signaling Technology).

Techniques: Functional Assay, Derivative Assay, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription, Western Blot, Control, Immunocytochemistry, Fluorescence, Membrane, Solvent, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: TRPV2 regulates cell fate in the human granulosa-like tumor cell line KGN: implications for granulosa cell tumors and cannabidiol

doi: 10.1186/s12964-026-02729-y

Figure Lengend Snippet: Altered proteome, increased cell size, proliferation rate, higher estradiol levels and macropinocytosis rate in TRPV2-deficient KGN cells. A Whole cell lysates from WT and TRPV2-deficient clone #4 subjected to mass spectrometry revealed massive changes of the proteome composition. A total of 993 proteins were upregulated, while 732 proteins were downregulated in TRPV2-deficient clones. As such, proteins related to regulation of actin cytoskeleton, but also sphingolipid and cholesterol metabolism are upregulated (a), whereas proteins associated with focal adhesion, pyroptosis and necroptotic process are downregulated upon depletion of TRPV2 (b). B The mean 3D diameter of trypsinized KGN cells is significantly increased in both TRPV2-deficient clones #4 and #13, compared to the WT ( n = 30, each). C Proliferation rate, determined by the incorporation of BrdU 48 h after cell seeding, revealed a significant increase upon depletion of TRPV2. Data are expressed as optical density at 380 nm in arbitrary units (a.U.; n = 14, each). D Impact of TRPV2 upon steroid production in KGN cells was assessed by subjecting supernatants derived from WT and TRPV2-deficient KGN cells to liquid chromatography-tandem mass spectrometry ( n = 3, each). Progesterone was only detected in supernatants from WT cells (n.d. - not detected), whereas the levels of estradiol were significantly higher in supernatants derived from TRPV2-deficient cells. E Importance of TRPV2 for macropinocytosis was assessed by measuring the uptake of 70 kDa FITC-dextran under 3D conditions. The TRPV2-deficient clone #4 engulfed significantly more than the other two cell types ( n = 8, each)

Article Snippet: Briefly, 10–15 μg protein were separated in a 10–15% SDS-PAGE and transferred to nitrocellulose membranes (AmershamTM ProtranTM, 0.45 μM; Thermo Fisher Scientific), which were incubated overnight at 4 °C with primary polyclonal rabbit IgG antibodies targeted either against TRPV2 (1:400; Atlas Antibodies), VDAC1 (Proteintech), cleaved Caspase 3 or 8 (clCasp3, #9664T, 1:1,000; clCasp8, #98134T, 1:1,000; Cell Signaling Technology).

Techniques: Mass Spectrometry, Clone Assay, Derivative Assay, Liquid Chromatography

Journal: Cell Communication and Signaling : CCS

Article Title: TRPV2 regulates cell fate in the human granulosa-like tumor cell line KGN: implications for granulosa cell tumors and cannabidiol

doi: 10.1186/s12964-026-02729-y

Figure Lengend Snippet: TRPV2-deficient KGN cells migrate faster. A Representative original micrographs from WT (a) and both TRPV2-deficient clones #4 (b) and #13 (c) captured at the beginning (upper panel; t = 0 h) and the end (lower panel; t = 24 h) of a 24 h scratch assay experiment. Scale bar 100 µM. B Mean motility as overall change in confluency (mean ± SEM; mean - solid line, SEM - dotted line) was analyzed using micrographs captured every 60 min for 24 h ( n = 18, each). Inset shows changes in confluency after 24 h (end-point) revealing significantly higher efficiency in gap closing in both TRPV2-deficient clones, compared to WT cells. C Single cell tracking was performed to determine the cumulative velocity on single cell level for each cell type ( n = 60, each), and revealed significantly higher cumulative velocity in both TRPV2-deficient KGN cells, compared to WT cells

Article Snippet: Briefly, 10–15 μg protein were separated in a 10–15% SDS-PAGE and transferred to nitrocellulose membranes (AmershamTM ProtranTM, 0.45 μM; Thermo Fisher Scientific), which were incubated overnight at 4 °C with primary polyclonal rabbit IgG antibodies targeted either against TRPV2 (1:400; Atlas Antibodies), VDAC1 (Proteintech), cleaved Caspase 3 or 8 (clCasp3, #9664T, 1:1,000; clCasp8, #98134T, 1:1,000; Cell Signaling Technology).

Techniques: Clone Assay, Wound Healing Assay, Single Cell Tracking, Single Cell

Journal: Cell Communication and Signaling : CCS

Article Title: TRPV2 regulates cell fate in the human granulosa-like tumor cell line KGN: implications for granulosa cell tumors and cannabidiol

doi: 10.1186/s12964-026-02729-y

Figure Lengend Snippet: Absence of TRPV2 makes KGN cell less vulnerable to CBD-induced depolarization and cell death. A Presence of CBD (30 µM; green) strongly increased the resting membrane potential of WT cells (light gray; n = 80 cells), whereas the TRPV2-deficient clones #4 (gray; n = 40 cells) and #13 (dark gray; n = 80 cells) were significantly less sensitive to such changes (statistical comparison the end point intensities), as demonstrated by means of membrane potential imaging experiments based on emission of the voltage sensitive fluorescence dye DiBAC4(3) (mean ± SEM; mean - black line, SEM - gray dotted line). Relative fluorescence intensity over time (normalized to starting point t 0 ), with representative live cell images of WT (top panel), and TRPV2-deficient clones #4 (middle panel) and #13 (bottom panel), respectively, displayed as pseudo-color images (black-purple – relatively hyperpolarized; yellow-white – relatively depolarized) at the start (a) and end of CBD presence within the dish (b). Scale bar 50 μm. B Consequences of application of the solvent control EtOH or different concentrations of CBD (1 µM, 5 µM, 10 µM, 15 µM and 30 µM) for 24 h on cell survival of WT cells ( n = 12) and TRPV2-deficient clones #4 ( n = 12) and #13 ( n = 11), respectively, were assessed by ATP assays. Results are shown as interpolated dose-effect curves based on all performed experiments. C Corresponding interpolated CBD IC 50 values for WT and TRPV2-deficient KGN cells revealed significantly higher concentrations needed to kill 50% of cells in absence of TRPV2. D Representative micrographs of WT (top) and TRPV2-deficient clones #4 (middle) and #13 (bottom) captured at the end of a 24 h treatment with either 7 µM CBD (a) or the solvent control EtOH (b), revealing the occurrence of cell death only in WT cells. Scale bar 100 μm

Article Snippet: Briefly, 10–15 μg protein were separated in a 10–15% SDS-PAGE and transferred to nitrocellulose membranes (AmershamTM ProtranTM, 0.45 μM; Thermo Fisher Scientific), which were incubated overnight at 4 °C with primary polyclonal rabbit IgG antibodies targeted either against TRPV2 (1:400; Atlas Antibodies), VDAC1 (Proteintech), cleaved Caspase 3 or 8 (clCasp3, #9664T, 1:1,000; clCasp8, #98134T, 1:1,000; Cell Signaling Technology).

Techniques: Membrane, Clone Assay, Comparison, Imaging, Fluorescence, Solvent, Control

Journal: Cell Communication and Signaling : CCS

Article Title: TRPV2 regulates cell fate in the human granulosa-like tumor cell line KGN: implications for granulosa cell tumors and cannabidiol

doi: 10.1186/s12964-026-02729-y

Figure Lengend Snippet: TRPV2 interactome in KGN cells and involvement of the mPTP in CBD-induced cell death. A TRPV2 interactome in KGN cells identified by immunoprecipitation followed by mass spectrometry. Top 20 of identified interactors (a) that can be categorized into three functional groups, i.e. regulation of intracellular transport, the regulation of apoptotic signaling pathway and cell growth, in accordance with a functional enrichment analysis. Note that VDAC1 and VDAC2 are among the top identified proteins and may be potential interactors. (b). B Impact of the mPTP formation blocker CysA (7 µM) on cytotoxic effects of CBD (7 µM) was assessed by means of microscopic monitoring (c & d) and ATP assays (e). Representative micrographs of WT (top) and TRPV2-deficient clones #4 (middle) and #13 (bottom) captured at the end of a 24 h treatment with either 7 µM CBD alone (c) or 7 µM CysA alongside with 7 µM CBD (d), revealing the occurrence of cell death only in WT cells whilst TRPV2-deficient clones appear healthy. CysA featured a protective effect on this CBD-induced cell death in WT KGN cells. Scale bar 100 μm. ATP assays quantitatively support this observation and confirmed the highly significant protective effect of CysA on CBD-induced cell death in all three cell types ( n = 15, each)

Article Snippet: Briefly, 10–15 μg protein were separated in a 10–15% SDS-PAGE and transferred to nitrocellulose membranes (AmershamTM ProtranTM, 0.45 μM; Thermo Fisher Scientific), which were incubated overnight at 4 °C with primary polyclonal rabbit IgG antibodies targeted either against TRPV2 (1:400; Atlas Antibodies), VDAC1 (Proteintech), cleaved Caspase 3 or 8 (clCasp3, #9664T, 1:1,000; clCasp8, #98134T, 1:1,000; Cell Signaling Technology).

Techniques: Immunoprecipitation, Mass Spectrometry, Functional Assay, Clone Assay

Journal: Molecular cell

Article Title: MRI is a DNA Damage Response Adaptor during Classical Non-Homologous End Joining

doi: 10.1016/j.molcel.2018.06.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-ATR , Novus Biologicals , Cat#NB100-323.

Techniques: Blocking Assay, Virus, Recombinant, Cell Isolation, Magnetic Beads, Mass Spectrometry, Sequencing, Magnetic Resonance Imaging, Plasmid Preparation, Software, High Content Screening, Flow Cytometry, Inverted Microscopy, Laser-Scanning Microscopy, Spectrophotometry, Irradiation

Journal: Molecular Biology of the Cell

Article Title: Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

doi: 10.1091/mbc.E21-03-0106

Figure Lengend Snippet: Cyclic mechanical stretch triggers transient adherens junction remodeling and actomyosin contraction. (A) Schematic illustration of stretch experiments. HUVECs were plated at confluency on silicon elastomers 24 h before stretch application, using negative pressure to deform the elastomer substrates (20%, 100 mHz), after which monolayers were analyzed at time points indicated. (B) Representative immunofluorescence images of VE-cadherin (VE-Cad) and pMLC2-stained HUVEC monolayers exposed to stretch. Note transient emergence of zipper-patterned adhesions and increased pMLC signal at 30 min of stretch. Scale bars 30 μm. (C) Close-up images of junctional rearrangements show reversibility of junctional zippering upon stretch. Scale bars 30 μm. (D) Quantification of AJ remodeling from VE-cadherin staining (left panel) and pMLC2 intensity (right panel). Mean ± SEM; n = 5 independent experiments; *** p = 0.0006, * p = 0.0203 (VE-cadherin) and) * p = 0.0168 and * p = 0.0071 (pMLC2), ANOVA, Dunnett’s. (E) Representative immunofluorescence images of α-18-stained HUVEC monolayers exposed to stretch. Note transient increase in α-18 intensity at 30 min of stretch. Scale bars 30 μm. (F) Quantification of α-18 catenin normalized to VE-cadherin intensity. Mean ± SD; n = 3 independent experiments; ** p = 0.0018, ANOVA, Dunnett’s.

Article Snippet: The following antibodies were used: VE-cadherin (Abcam AB33168; 1:400), β-catenin (Santa Cruz Biotechnology; sc-7199; 1:500) Phospho-Myosin Light Chain 2 (Thr18/Ser19; Cell Signaling; 3674; 1:200), vinculin (Millipore MAB 3574, 1:500), α18 ( Yonemura et al. , 2010 ), 1:8000), HA-Tag (Cell Signaling 2367; 1:1000) streptavidin (ThermoFisher Scientific MA1-20010; 1:1000), and Alexa Fluor 488, 568, and 647 conjugated secondary antibodies (Invitrogen; 1:400).

Techniques: Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

doi: 10.1091/mbc.E21-03-0106

Figure Lengend Snippet: AJ remodeling and reinforcement is associated with subtle changes in VE-cadherin interactome. (A) Schematic illustration of VE-cadherin (VE-Cad)–BioID2 pulldown and mass spectrometry to identify stretch-dependent interactome changes. (B) Representative immunofluorescence images showing localization of HA-tagged VE-cadherin–BioID2 at AJs and enrichment of streptavidin as a result of biotin binding at this site. Scale bars 30 μm. (C) Volcano plot of protein enrichment in VE-cadherin–BioID2 compared with BioID2 control. Note enrichment of VE-cadherin itself ( CDH5 ) along with α- and β-catenin ( CTNNA1, CTNNB1 ), indicative of successful enrichment of AJ components. Also highlighted are cytoskeletal and adhesion remodelers. Dotted line marks padj cutoff of 0.05, moderated t test/Benjamini Hochberg. (D) GO term enrichment analysis of proteins significantly enriched in VE-cadherin–BioID2 pulldowns.

Article Snippet: The following antibodies were used: VE-cadherin (Abcam AB33168; 1:400), β-catenin (Santa Cruz Biotechnology; sc-7199; 1:500) Phospho-Myosin Light Chain 2 (Thr18/Ser19; Cell Signaling; 3674; 1:200), vinculin (Millipore MAB 3574, 1:500), α18 ( Yonemura et al. , 2010 ), 1:8000), HA-Tag (Cell Signaling 2367; 1:1000) streptavidin (ThermoFisher Scientific MA1-20010; 1:1000), and Alexa Fluor 488, 568, and 647 conjugated secondary antibodies (Invitrogen; 1:400).

Techniques: Mass Spectrometry, Immunofluorescence, Binding Assay, Protein Enrichment, Control

Journal: Molecular Biology of the Cell

Article Title: Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

doi: 10.1091/mbc.E21-03-0106

Figure Lengend Snippet: Stretch triggers transient calcium signaling and activation of Rho and GTPases to remodel junctions. (A) Quantification of RhoA activity over time shows peak of Rho activation at 5 min of stretch (mean ± SD; n = 3 independent experiments; * p = 0.0461, ANOVA with Dunnett’s). (B) Quantification of Rac1 activity over time shows peak of Rac activation at 30 min of stretch (mean ±SD; n = 4 independent experiments; * p = 0.0163, ANOVA with Dunnett’s). (C) Representative immunofluorescence images and quantification of AJ remodeling from β-catenin and F-actin (phalloidin)-stained HUVEC monolayers exposed to stretch in the presence of either Y-27632 or IPA3. Note prevention of adhesion zippering in Y-27632-treated cells and attenuated junction restoration in IPA3-treated cells. Scale bars 30 μm (mean ± SD; n = 3 independent experiments; *** p < 0.0001,** p = 0.0015, ANOVA with Dunnett’s). (D, E) Representative images, D, and quantification, E, of calcium imaging with Fluo-4-AM shows a transient increase in intracellular calcium at 30 min of stretch (mean ± SD; n = 3 independent experiments; * p = 0.0106, ANOVA with Dunnett’s; scale bars 30 μm). (F) Representative immunofluorescence images of confluent, transiently VE-cadherin-Apple transfected HUVEC monolayers showing remodeling of VE-cadherin junctions in response to ionophore application. Scale bars 30 μm.

Article Snippet: The following antibodies were used: VE-cadherin (Abcam AB33168; 1:400), β-catenin (Santa Cruz Biotechnology; sc-7199; 1:500) Phospho-Myosin Light Chain 2 (Thr18/Ser19; Cell Signaling; 3674; 1:200), vinculin (Millipore MAB 3574, 1:500), α18 ( Yonemura et al. , 2010 ), 1:8000), HA-Tag (Cell Signaling 2367; 1:1000) streptavidin (ThermoFisher Scientific MA1-20010; 1:1000), and Alexa Fluor 488, 568, and 647 conjugated secondary antibodies (Invitrogen; 1:400).

Techniques: Activation Assay, Activity Assay, Immunofluorescence, Staining, Imaging, Transfection

Journal: Molecular Biology of the Cell

Article Title: Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

doi: 10.1091/mbc.E21-03-0106

Figure Lengend Snippet: Stretch reduces Piezo1 levels to attenuate calcium signaling. (A, B) Representative immunofluorescence images, A, and quantification, B, of AJ remodeling from β-catenin stained junctions show prevention of junctional remodeling in the presence of GsMTx4 at 30 min of stretch (mean ± SD; n = 3 independent experiments; ** p = 0.007, ANOVA with Dunnett’s; scale bars 30 μm). (C, D) Representative immunofluorescence images, C, and quantification, D, of pMLC2 and AJ remodeling from VE-cadherin stained HUVECs with control or Piezo1 siRNA cells show attenuation of pMLC2 elevation and junctional remodeling in Piezo1-depleted at 30 min of stretch (mean ± SD; n = 3 independent experiments; ** p = 0.0017 for remodeled AJ and * p = 0.0219,** p = 0.0014 for pMLC2 intensity, ANOVA with Dunnett’s; scale bars 30 μm). (E, F) Representative Western blot, E, and quantification, F, show reduction of Piezo1 protein levels at 30 min of stretch (mean ± SD; n = 3 independent experiments; * p = 0.025, ANOVA with Dunnett’s).

Article Snippet: The following antibodies were used: VE-cadherin (Abcam AB33168; 1:400), β-catenin (Santa Cruz Biotechnology; sc-7199; 1:500) Phospho-Myosin Light Chain 2 (Thr18/Ser19; Cell Signaling; 3674; 1:200), vinculin (Millipore MAB 3574, 1:500), α18 ( Yonemura et al. , 2010 ), 1:8000), HA-Tag (Cell Signaling 2367; 1:1000) streptavidin (ThermoFisher Scientific MA1-20010; 1:1000), and Alexa Fluor 488, 568, and 647 conjugated secondary antibodies (Invitrogen; 1:400).

Techniques: Immunofluorescence, Staining, Control, Western Blot

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Six marker FFPE panel for imaging mass spectrometry (Hyperion).

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Marker, Imaging, Mass Spectrometry

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Analysis of squamous cell carcinoma (SCC) primary tumors via immunohistochemistry, all tissues were stained for endoglin expression (brown). The black arrows indicate endothelial endoglin expression. The white arrows indicate epithelial endoglin expression. (A) Representative images of ESCC ( n = 9). (B) Representative images of HNSCC ( n = 5). (C) Representative images of VSCC ( n = 7). Images taken at 100x (left) and 200x (right).

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Analysis of HNSCC via imaging mass spectrometry (Hyperion) with a six marker panels. (A) Six images, each depicting the expression of the corresponding marker. (B) A merged image combining pan-cytokeratin, endoglin, and p53 expression. The white arrow indicates cells that co-express pan-cytokeratin and endoglin, which are negative for p53. (C) A merged image combining pan-cytokeratin, endoglin, and CD68 expression. The white arrow indicates cells that co-express pan-cytokeratin and endoglin, which are negative for CD68.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Imaging, Mass Spectrometry, Marker, Expressing

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Analysis of ESCC via imaging mass spectrometry (Hyperion) with a six marker panels. (A) Six images, each depicting the expression of the corresponding marker. (B) A merged image combining pan-cytokeratin, endoglin, and p53 expression. The white arrows indicate cells that co-express pan-cytokeratin and endoglin, which are negative for p53. (C) A merged image combining pan-cytokeratin, endoglin, and CD68 expression. The white arrow indicates cells that co-express pan-cytokeratin and endoglin, which are negative for CD68. The blue arrow indicates cells that co-express pan-cytokeratin, endoglin, and CD68.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Imaging, Mass Spectrometry, Marker, Expressing

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Analysis of VSCC via imaging mass spectrometry (Hyperion) with a six marker panel. (A) Six images, each depicting the expression of the corresponding marker. (B) A merged image combining pan-cytokeratin, endoglin, and p53 expression. The white arrows indicate cells that co-express pan-cytokeratin and endoglin. (C) A merged image combining pan-cytokeratin, endoglin, and CD68 expression. The white arrow indicates cells that co-express pan-cytokeratin and endoglin, which are negative for CD68. The blue arrow indicates cells that co-express pan-cytokeratin, endoglin, and CD68.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Imaging, Mass Spectrometry, Marker, Expressing

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: The expression of endoglin by SCC cell lines. Expression by 10 ESCC cell lines, where endoglin expression by TE01 ( p < 0.0001) and TE15 ( p < 0.003) significantly differ from all other cell lines (A) , OSC-19 and FaDu show significantly different endoglin expression ( p = 0.0002) (B) , as is also detected in the three VSCC cell lines (* p = 0.0123, ** p = 0.0025, *** p = 0.0001) with different morphologies (conventional—VC415-C and VC704; spindle—VC415-S) (C) . Endoglin protein levels were determined via western blot (D) and ELISA. Endoglin protein expression by TE01 significantly differs from all other cell lines—( p ≤ 0.0018) (E) . Image for western blot analysis is a representative image of n = 2–3 independent experiments.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Analysis of ALK expression by SCC cell lines, determined via qPCR. Gene expression for ALK1—ALK7 by TE10—endoglin low (A) , TE11—endoglin low (B) , TE01—endoglin high (C) , OSC-19—endoglin positive (D) , FaDu–endoglin high (E) , VC415-C—endoglin low, and VC415-S—endoglin high; *** p = 0.000581 (F) . The effect of endoglin overexpression (OE) on ALK expression was analyzed for TE10; *** p =0.000126; ** p =0.003291 (G) and TE11 (H) , as well as the effect of endoglin knockout (KO) in TE01 (I) .

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Expressing, Gene Expression, Over Expression, Knock-Out

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: SCC cells were stimulated with either BMP-6 (TE10 and TE11 only), BMP-9 or TGF-β and the level of phosphorylated SMAD1 and SMAD2 (pSMAD1 and pSMAD2) was determined via western blot (A–C) . The amount of soluble endoglin in the medium of TE10 and TE11 was determined via ELISA (D,E) . Stimulation of OSC-19 (F) , FaDu (G) , VC415-C (H) , and VC415-S (I) with BMP-9/TGF-β/TRC105 was performed, and the levels of pSMAD1 and pSMAD2 were determined via western blot. Western blot images are representative of n = 2–3 per experiment.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: SCC cell lines were stimulated with BMP-9 or TGF-β and the proliferation of TE10 (A) , TE11 (C) , TE01 (E) , and VC415-S (G) cells was measured via a MTS assay. To assess the effects of endoglin on cell proliferation, MTS assays were performed on endoglin overexpressing (OE) TE10 (B) and TE11 (D) cells. The effects of endoglin knockout (KO) in TE01 (F) and endoglin knockdown (KD) in VC415-S (H) were also assessed via MTS. n = 2–3 for each experiment.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: MTS Assay, Knock-Out, Knockdown

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: SCC cell lines were stimulated with BMP-9 or TGF-β and the migration of TE10 (A) , TE11 (C) , TE01 (E) , VC415-S (G) , OSC-19 (I) , and FaDu (J) cells was measured via a wound healing assay. To assess the effects of endoglin on cell migration, wound healing assays were performed on endoglin overexpressing (OE) TE10 (B) and TE11 (D) cells. The effects of endoglin knockout (KO) in TE01 (F) and endoglin knockdown (KD) in VC415-S (H) were also assessed. Finally, the effects of TRC105 on cell migration was assessed in OSC-19 (I) and FaDu cells (J) . n = 2–3 for each experiment.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Migration, Wound Healing Assay, Knock-Out, Knockdown

Journal: Frontiers in Medicine

Article Title: Endoglin and squamous cell carcinomas

doi: 10.3389/fmed.2023.1112573

Figure Lengend Snippet: Summary table of the relationship between (altered) endoglin expression, BMP-9 signaling, TGF-β signaling, SCC cell migration, and SCC cell proliferation.

Article Snippet: Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature.

Techniques: Expressing, Migration