Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: Summary of sero-negative, acute, and convalescent-phase of COVID-19 patients from which the plasma ACE2 + EVs and RBD-IgG levels were measured.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Sampling

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of the cell-based neutralization assay. b Representative flow profiles showing the percentage (fluorescence mean intensity) of RBD-AF647 binding (at 16 and 3.3 nmol/L) to ACE2 + HEK-293 cells, inhibited by rhACE2 and ACE2 + EVs (evACE2) isolated from HEK-293 and HeLa cells (HEK-EV1 and HeLa-EV2, respectively) whereas ACE2 − EVs (evCon) had no neutralization effects (no RBD in black, PBS in dark blue, rhACE2 in orange, evCon in light blue, and evACE2 in green). c IC 50 of rhACE2 (orange line) and ACE2 in the EVs from ACE2 + HEK (ev1ACE2) and HeLa (ev2ACE2) cells (green lines) on 16 nM RBD-host cell binding (%). GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. d IC 50 of evACE2, ev1 from HEK and ev2 from HeLa cells (green lines), and rhACE2 (orange line) neutralizing infections by wild-type (WT) S + pseudotyped SARS-CoV-2. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. e IC 50 (nM) of ACE2 in ev1ACE2 (HEK) (green line) and rhACE2 (orange line) upon wild-type SARS-CoV-2 infection. GraphPad Prism 9.0.2 was used to calculate the IC 50 with three biological replicates. Data are presented as mean values ± SD. f Distinct effects of ACE2 + EVs (green lines) and ACE2 − control EVs (light blue line) on inhibiting Vero-6 cell death caused by SARS-CoV-2. N = 2 experiments with three biological replicates each. Data are presented as mean values ± SD. g The IC 50 of ev1ACE2 (HEK) neutralizing infections by pseudotyped SARS-CoV-2 expressing WT (black), B.1.1.7 (α) variant (red), B1.351 (β) variant (dark blue) and B.1.617.2 (δ) (light green) S protein. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates each. Data are presented as mean values ± SD. h Effects of ev1ACE2 (HEK) on protecting Vero-6 cell viability against infections of SARS-CoV-2 WT (black), B.1.1.7 (α) variant (red) and B1.351 (β) variant (dark blue) ( n = 3 biological replicates). Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Neutralization, Fluorescence, Binding Assay, Isolation, Infection, Control, Expressing, Variant Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of plasma EV ultracentrifugation and RBD-bead based depletion. b Cryo-EM images of human EV pellets isolated from acute phase COVID-19 plasma (bar = 100 nm). c Immunoblots of plasma EV pellets (sero-negative and COVID-19 acute phase patients CBB-005 and -013) for ACE2 and loading control of protein staining with Ponceau). Laemmli buffer was used for lysis ( N = 1 experiment). d ACE2 + EV pellets from acute phase patients 007, 008, 009, 012, and 013 (CBB) ( n = 2 biological replicates each) blocked SARS-CoV-2 infection-induced death of Vero-6 cells whereas the sero-negative control ( n = 2 biological replicates) and CBB-005 (no detectable ACE2) ( n = 2 biological replicates) did not show neutralization effects. One-tail t test, **** p = 2.24E−08 shown as compared to sero-negative. e , f Levels of ACE2 + EV counts ( n = 3 biological replicates) in plasma EVs (green) and bead-depleted EVs (light blue). One-tail paired t test, * p = 0.011 and ** p = 0.0063 (data are presented as mean values ± SD) ( e ) and altered neutralization effects on RBD–host cell binding ( f ) of the COVID-19 plasma EV pellets prior to and after RBD-bead depletion (convalescent phase CSB-012 and -024; acute phase CBB-008, 009, and 013). One-tail paired t test **** p = 5.11E−05.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Cryo-EM Sample Prep, Isolation, Western Blot, Control, Staining, Lysis, Infection, Negative Control, Neutralization, Binding Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Probability of severe disease-free survival in B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice receiving SARS-CoV-2 infection (10,000 pfu) and intranasal EVs (130 µg as measured on Nanodrop) per mouse (evCon in light blue and evACE2 in green). Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests **** p = 2.27E−07. b Viral loads in mouse lungs on day 5/6 after receiving SARS-CoV-2 infection and administration of evCon ( N = 5 mice) (light blue) or evACE2 ( N = 10 mice) (green). T -test–nonparametric-one tailed, * p = 0.013. Data are presented as mean values ± SD. c . Representative H&E images of mouse lung sections at day 5 or 6 post virus inoculation and EV treatment (evCon and evACE2) intranasally. d , e Acute and chronic inflammation scores ( d ), and alveolar hemorrhage and necrosis scores ( e ) in mouse lungs on day 5/6 after receiving evCon ( N = 5 mice) (light blue) or evACE2 ( N = 7 mice) (green). T -test–nonparametric-one tailed, ** p = 0.005, ** p = 0.003 and *** p = 0.0004. Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Infection, One-tailed Test, Virus

Journal: Cell Reports

Article Title: Molecular Deconvolution Platform to Establish Disease Mechanisms by Surveying GPCR Signaling

doi: 10.1016/j.celrep.2018.06.080

Figure Lengend Snippet:

Article Snippet: Human: HEK293T/17 , ATCC , CRL11268.

Techniques: Recombinant, Modification, Saline, Protease Inhibitor, Luciferase, One Step RT-PCR, Knock-Out, Plasmid Preparation, CRISPR, Software

Journal: Chinese Neurosurgical Journal

Article Title: Downregulation of lncRNA-HOXA11-AS modulates proliferation and stemness in Glioma cells

doi: 10.1186/s41016-017-0091-6

Figure Lengend Snippet: Fig. 2 HOXA11-AS regulates glioma stemness in vitro. a mRNA expression of HOXA11-AS genes was determined in GBM cell lines and normal epithelial cell cultures by qRT-PCR. Expression levels are shown relative to the expression in HEK293 cells, which was used as a control and set as 1. b qRT-PCR was used to detect HOXA11-AS expression of U87MG cells with lenti-HOXA11-AS knockdown. c qPCR panels represent HOXA11-AS expression in U87MG cells infected with lentivirus-HOXA11-AS overexpression and incubated with different concentrations of doxycyclin (dox) in the culture medium for 2 days. d and e HOXA11-AS expressing or silenced U87MG cells (1 × 103) were seeded in ultra-low attachment plate and allowed to grow for 10 days in stem cell culture medium. Images were taken under light microscope and the number of spheres (size >50 μm) was counted. (**p < 0.01; ****p < 0.0001)

Article Snippet: Cell culture and viral infection Human GBM cell lines U87MG, LN229, U251 and human embryonic kidney 293 (HEK293) cells were purchased from ATCC (the American Type Culture Collection, Manassas, VA, USA).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Control, Knockdown, Infection, Over Expression, Incubation, Stem Cell Culture, Light Microscopy