Journal: Science Advances

Article Title: LIMCH1-enriched extracellular vesicles promote vascular permeability in early-onset preeclampsia

doi: 10.1126/sciadv.aeb8806

Figure Lengend Snippet: ( A ) Generation of LIMCH1-OE JAR cells. LIMCH1 expression was measured by RT-qPCR with glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) as an internal control. *** P < 0.001, Student’s t test. ( B ) Immunoblot analysis of LIMCH1 and β-actin in control, LIMCH1-OE JAR cells, and their derived EVs. ( C ) Cell viability of JAR-control and JAR-LIMCH1-OE cells. Data are shown as means ± SD. ( D ) Size distribution of JAR-derived sEVs using NTA. ( E ) Morphology of JAR-sEV detected using TEM. Scale bar, 200 nm. ( F ) Immunoblot analysis of tetraspanin markers (CD9, CD63, and CD81) in JAR cell lysates and sEVs. ( G ) Schematic representation of the experimental protocol for EV uptake into HUVECs. CellMask Green–labeled EVs were added to HUVECs and observed after 5 h (hours). ( H ) Representative confocal microscopy images. EVs were labeled with CellMask Green, and cell membrane and nuclei were stained with CellMask Deep Red (red) and Hoechst (blue), respectively. Scale bar, 10 μm. Right graph shows quantitative analysis using NIS-Elements AR. Data are shown as means ± SD. ns, no significance; Student’s t test. Biological n = 3 per condition. Images represent cropped regions of fig. S5. ( I and J ) In vitro permeability assay using 40-kDa (I) and 70-kDa (J) FITC-dextran. HUVECs cultured on transwell inserts were treated with 6 μg of Control-EVs or LIMCH1-EVs. FITC-dextran was added after 24 hours, fluorescence in the lower chamber was measured as the optical density (OD), and OD after 4 hours was compared. Data are shown as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test. ( K ) TEER measurements. Data are shown as means ± SD. * P < 0.05, Student’s t test. Data are representative of at least three independent experiments [(I) to (K)].

Article Snippet: To generate CellMask green-labeled PBS, 1 μl of CellMask Green plasma membrane stain (Invitrogen) was added to 200 μl of PBS and incubated for 30 min at 37°C and transferred to a 1.5-ml microcentrifuge tube.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Derivative Assay, Labeling, Confocal Microscopy, Membrane, Staining, In Vitro, Permeability, Cell Culture, Fluorescence

Journal: Science Advances

Article Title: LIMCH1-enriched extracellular vesicles promote vascular permeability in early-onset preeclampsia

doi: 10.1126/sciadv.aeb8806

Figure Lengend Snippet: ( A ) Schematic representative protocol of the Evans blue permeability assay in vivo. ICR mice were administered 100 μl PBS containing 10 μg of Control-EVs ( n = 6) or LIMCH1-EVs ( n = 6) retro-orbitally. Twenty-four hours later, 100 μl of 2% Evans blue dye was injected retro-orbitally. Lung and brain tissues were collected 1 hour after injection of Evans blue dye. ( B ) Images of the mice. White arrow shows a mouse several minutes after Evans blue injection. ( C ) Representative image of the lungs collected from Control-EV–injected and LIMCH1-EV–injected mice. Scale bar, 1 mm. Graphs showing the quantitative results of Evans blue permeability assay (left, nonpregnant mice; right: pregnant mice). Data are shown as means ± SD. * P < 0.05, Student’s t test. ( D ) Representative image of the brain collected from Control-EV–injected and LIMCH1-EV–injected mice. Scale bar, 1 mm. Graphs showing the quantitative results of Evans blue permeability assay (left, nonpregnant mice; right, pregnant mice). Data are shown as means ± SD. ns, no significance; Student’s t test. ( E ) Representative images using confocal microscopy. EVs were labeled with CellMask Green, and ICR mice were administered CellMask Green–labeled PBS ( n = 1) or CellMask Green–labeled 10 μg of JAR EVs ( n = 3). Nuclei were counterstained blue with DAPI. White arrows indicate cells with abundant EV uptake. Scale bar, 10 μm. The images shown here represent magnified regions of the corresponding images presented in fig. S9A. Bottom right graph shows a quantitative result of NIS-Elements AR Analysis program. Data are shown as means ± SD. ** P < 0.01, Student’s t test.

Article Snippet: To generate CellMask green-labeled PBS, 1 μl of CellMask Green plasma membrane stain (Invitrogen) was added to 200 μl of PBS and incubated for 30 min at 37°C and transferred to a 1.5-ml microcentrifuge tube.

Techniques: Permeability, In Vivo, Control, Injection, Confocal Microscopy, Labeling