Protein to lipid ratios of EV subpopulations.

<p>Protein to lipid ratios are presented for APOs, MVs, and EXOs isolated from MH-S (A), THP-1 (B), and BV-2 (C) cell lines (data represent ≥ 3 independent experiments for each EV type from all cell lines). Combined protein to lipid ratios obtained using EVs derived from MH-S, THP-1, BV-2, Jurkat, U937 as well as from human blood plasma are shown (D) (results of ≥ 12 independent experiments each EV type). Combined protein (E) and lipid concentrations (F) for the above cell line derived and blood plasma derived EVs are also shown. Mean values are represented by horizontal lines, and standard error means (SEMs) are indicated by error bars. The mean values ± SEM of lipid and protein concentrations (μg/mL) of conditioned media are reported below each respective EV subpopulation.</p

Design and Synthesis of Sulfamoyl Benzoic Acid Analogues with Subnanomolar Agonist Activity Specific to the LPA₂ Receptor

Lysophosphatidic acid (LPA) is a growth factor-like mediator and a ligand for multiple GPCR. The LPA₂ GPCR mediates antiapoptotic and mucosal barrier-protective effects in the gut. We synthesized sulfamoyl benzoic acid (SBA) analogues that are the first specific agonists of LPA₂, some with subnanomolar activity. We developed an experimental SAR that is supported and rationalized by computational docking analysis of the SBA compounds into the LPA₂ ligand-binding pocket

Characterization of EV subpopulation size and morphology.

<p>Apoptotic bodies (APO), microvesicles (MV) and exosomes (EXO) were isolated from conditioned tissue culture supernatant of BV-2 cells. Size distributions of the different subpopulations were assessed by tunable resistive pulse sensing (qNano) using three different membranes (NP200, NP400, and NP2000). Obtained particle concentrations were merged into single histograms for each EV subpopulation (left panels). Electron microscopic images of respective EV subpopulation pellets are shown (right panels).</p

Flow cytometric characterization of EV subpopulations.

<p>EV subpopulations were isolated from BV-2 cells, and were analyzed along with the releasing cells either directly (MVs and APOs) or after coupling to latex beads (EXOs) by flow cytometry using annexin V FITC, as well as anti-CD9 FITC, anti-CD63 PE and anti-cholesterol CF488-conjugated antibodies, and Alexa Fluor647-conjugated cholera toxin (CTX) (all marked with thick black lines), and were compared to respective negative controls (thin black lines). Isotype controls were used for samples stained with fluorochrome-conjugated antibodies, whereas autofluorescence was detected in the absence of either annexin V or CTX. Images and figures are representatives of at least three independent experiments.</p

Spectral ratiometric determination of EV membrane lipid order.

<p>A shows the quantitative assessment of membrane lipid order in subpopulations of EVs secreted by BV-2 cells is shown (≥3 independent experiments each EV type). Ratiometric measurements of the fluorescent intensities at 560–600nm and at >660nm were carried out by confocal microscopy, and results are expressed as general polarization (GP) values. The higher GP value reflects a higher membrane lipid order. EXOs showed the highest order, while APOs and MVs were characterized by partially overlapping, intermediate order. B shows the flow cytometric determination of di-4-ANEPPDHQ staining of EV subpopulations secreted by BV-2 cells. Representative results of one out of n = 3 independent experiments. Left panels represent the fluorescence at 585±21 nm, while right panels represent fluorescence at 616±12 nm of unstained and stained vesicles and beads (thin and thick lines, respectively). Geometric mean fluorescent intensities (MFI) of unstained and stained APOs, MVs, EXOs coupled to latex beads, and beads without EXOs are shown (regular and bold text, respectively).</p

Protein to lipid ratio as a quality control parameter of EV preparations.

<p>A-C show results of 4 independent experiments where there were visible pellets for both the 20000g (MV) and 100,000g (“EXO”) preparations. While the normal protein to lipid ratio of MVs reflected a true vesicular pellet as also demonstrated by electron microscopy (B), a strongly elevated protein to lipid ratio suggested the absence of EXOs as was later confirmed by electron microscopy (C).</p

Doxycycline Inducible Kruppel-Like Factor 4 Lentiviral Vector Mediates Mesenchymal to Epithelial Transition in Ovarian Cancer Cells

<div><p>Ovarian cancer presents therapeutic challenges due to its typically late detection, aggressive metastasis, and therapeutic resistance. The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in human cancers as a tumor suppressor or oncogene, although its role depends greatly on the cellular context. The role of KLF4 in ovarian cancer has not been elucidated in mechanistic detail. In this study, we investigated the role of KLF4 in ovarian cancer cells by transducing the ovarian cancer cell lines SKOV3 and OVCAR3 with a doxycycline-inducible KLF4 lentiviral vector. Overexpression of KLF4 reduced cell proliferation, migration, and invasion. The epithelial cell marker gene E-cadherin was significantly upregulated, whereas the mesenchymal cell marker genes vimentin, twist1and snail2 (slug) were downregulated in both KLF4-expressing SKOV3 and OVCAR3 cells. KLF4 inhibited the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Taken together, our data demonstrate that KLF4 functions as a tumor suppressor gene in ovarian cancer cells by inhibiting TGFβ-induced EMT.</p></div

KLF4 promotes mesenchymal-epithelial cell transition.

<p><b>A. B</b>. Western blots were performed in KLF4- and EGFP-transduced SKOV3 (<b>A</b>) and OVCAR3 cells (<b>B</b>) with or without Dox induction. E-cadherin expression was significantly upregulated (***<i>p</i><0.001), whereas vimentin (**<i>p</i><0.01) and snail2 (***<i>p</i><0.001) were downregulated in KLF4- overexpressing SKOV3 cells compared to control cells (<b>A</b>). E-cadherin (**<i>p</i><0.01) was upregulated, whereas vimentin (*<i>p</i><0.05) and snail2 (***<i>p</i><0.001) were downregulated in KLF4-overexpressing OVCAR3 cells compared to Dox-treated control cells (<b>B</b>). E-cadherin (<b>C</b>) and vimentin (<b>D</b>) were immunostained in cellular membranes in KLF4-expressing and control SKOV3 cells. <b>E</b>. Snail2 was stained in cell nuclei in KLF4-expressing and control SKOV3 cells. <b>F</b>. KLF4 binding to the promoter of E-cadherin in SKOV3 cells was examined by chromatin immunoprecipitation using KLF4 antibody and detected by real-time PCR using E-cadherin-specific primers. The ChIP-enriched DNA levels were normalized to input DNA, followed by subtraction of non-specific binding determined by control IgG (***<i>p</i><0.001).</p

KLF4 reduces ovarian cancer cell invasion.

<p><b>A, B</b>. Cell invasion assay was performed using Matrigel-coated transwell plates for SKOV3 (<b>A</b>) and OVCAR3 cells (<b>B</b>). The invasion rate was significantly reduced in KLF4-overexpressing cells compared to that in Dox-treated control cells from both SKOV3 and OVCAR3 cells (**<i>p</i><0.01). Data were collected from three separate experiments and analyzed using Student <i>t</i>-tests.</p

KLF4 inhibits TGFβ-induced EMT in ovarian cancer cells.

<p>Ovarian cancer cell lines SKOV3 (<b>A</b>) and OVCAR3 (<b>B</b>) transduced with lentiviral KLF4 overexpression and control vector were treated with TGFβ for 48 h, and the protein expressions of EMT-associated marker genes E-cadherin, snail2 and vimentin were examined using Western blot. Significant differences were compared between KLF4 expressing and control group (**p<0.05, ***p<0.001).</p