Journal: Frontiers in Cell and Developmental Biology

Article Title: SCA-1/Ly6A Mesodermal Skeletal Progenitor Subpopulations Reveal Differential Commitment of Early Limb Bud Cells

doi: 10.3389/fcell.2021.656999

Figure Lengend Snippet: The morphogenetic capacity of Sca-1 limb bud cells in the recombinant limb model. (A) Schematic representation of the experimental design of recombinant limbs experiments. Recombinant limbs (RLs) were performed with non-electroporated (NE), sham (GFP), and Sca-1 electroporated limb bud cells (e-Sca-1 + ). Mesodermal hindlimb cells and ectoderms were obtained from 22HH embryos. Electroporated (GFP or e-Sca-1) and NE recombinant limbs were implanted in 22HH chick embryos and collected after 2 (a–f) or 6 (g–l) days. (B) Alcian blue stain to evidence skeletal elements (a,c,e,g,i,k) . Sagittal slices of RL after Alcian blue staining stained with hematoxylin and eosin (b,d,f,h,j,l) . Square delimits the magnification for b′,d′,f′. Scale bars are representative for all conditions representing 100 μm. qRT-PCR of chondrogenic (C) , tenogenic (D) , and tendon cell fate regulating genes (E) of the e-Sca + 48h RLs relative to the GFP-RLs is shown (set to 1.0, dashed red line). GFP-RLs and NE-RLs showed no statistical differences between all genes (data no show). Data represent three independent experiments. Statistical significance was set as follows: ** p < 0.005, * p < 0.05.

Article Snippet: Immediately after, 5 × 10 6 cells were resuspended in Multiporator Electroporation Buffer Isosmolar (Eppendorf, Hamburg, Germany, cat. no. EMP4308070510), transferred into cuvettes, and electroporated with 1.5 μg of Sca-1 -GFP (Origene, cat. no. MG200790) or GFP (sham) plasmid per million cells using an Eppendorf Multiporator System.

Techniques: Recombinant, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) Levels of Ly6E, GILT and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at the indicated time after the addition of tet. Intracellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). **, p <0.001 compared to the control cells expressing CAT.

Article Snippet: The cDNA molecules of ADAP2 and LY6E were purchased from OriGene (Cat. No. RC207501 and RC211373, respectively ) and cloned into pcDNA5/FRT-derived vector as described previously ( ).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of intracellular LY6E expression was determined by Western blot using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations (n = 4). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red). Cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: The cDNA molecules of ADAP2 and LY6E were purchased from OriGene (Cat. No. RC207501 and RC211373, respectively ) and cloned into pcDNA5/FRT-derived vector as described previously ( ).

Techniques: Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Infection, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is conducted and “three finger-fold” structure is highlighted with black box. The conserved L36 as well as GPI anchor and N99 glycosylation sites are indicated (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT, wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. Intracellular LY6E expression were detected by a Western blot assay. β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4).

Article Snippet: The cDNA molecules of ADAP2 and LY6E were purchased from OriGene (Cat. No. RC207501 and RC211373, respectively ) and cloned into pcDNA5/FRT-derived vector as described previously ( ).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: Flp-In T-Rex 293-derived cell line expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48hpi. ( A ) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1). Error bars indicate the standard deviation (n=4). ( B ) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation (n=4). **, p <0.001, comparing to cells transfected with pCAGGS vector.

Article Snippet: The cDNA molecules of ADAP2 and LY6E were purchased from OriGene (Cat. No. RC207501 and RC211373, respectively ) and cloned into pcDNA5/FRT-derived vector as described previously ( ).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Standard Deviation, Activity Assay

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: Flp-In T-Rex 293-derived cell line expressing IFITM3 ( A ) or LY6E ( B ) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1μM AmphoB. Luciferase activity was measured at 48 hr post-infection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). **, p <0.001, compared to mock treatment.

Article Snippet: The cDNA molecules of ADAP2 and LY6E were purchased from OriGene (Cat. No. RC207501 and RC211373, respectively ) and cloned into pcDNA5/FRT-derived vector as described previously ( ).

Techniques: Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: scRNA-seq analyses revealed Sca1 + cells repopulate ECs in mouse femoral artery after injury (A) Normal femoral arteries (NC) and injured femoral arteries were digested and analyzed by scRNA-seq after 2 (FAI2W) or 4 (FAI4W) weeks. Umap plot showed visualization of unsupervised clustering of all groups. (B) Dot plot of canonical cell markers identified EC cluster ( Kdr , Flt1 , Cdh5 , Pecam1 , and Nos3 ) and SPC cluster ( Cd34 , Ly6a , and Pdgfra ). (C) Bar plot showed the constitution of Ly6a (Sca1) + or Ly6a − cells among ECs of each group. (D) Differentially expressed genes between Ly6a + and Ly6a − cells were identified and analyzed using GO enrichment. The top 10 enriched biological process terms were presented. (E) Trajectory analysis of the differentiation process from SPCs to ECs; the color change from green to blue indicates the cell’s progression from a primitive to a more developed state. (F) The expression levels of canonical EC markers and SPC markers are shown alongside the differentiation trajectory. See also Figure S1 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Expressing

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Vascular Sca1 + cells exhibit endothelial differentiation potential in artery injury and repair (A) Immunofluorescence staining of healthy femoral artery for CD31 and Sca1 + lineage marker tdTomato (tdT) using Sca1-CreER T2 ;Rosa26-tdTomato mouse model. The right panel showed magnification of the boxed region. (B) Immunofluorescence staining of injured femoral artery for CD31 and tdTomato (Sca1 + lineage). The right panel and the panel below showed magnification of the boxed regions. For merge images, scale bars, 100 μm. For magnification images, scale bars, 20 μm. (C) Quantification of the percentage of CD31 + tdT + cells in CD31 + cells of healthy femoral artery (HFA) and injured femoral artery (IFA). Data represent mean ± SEM, n = 6 in each group. p value was specified in the graph. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values. See also Figure S2 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Marker, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Vascular Sca1 + cells differentiate into ECs in vein graft (A) Immunofluorescence staining of normal vena cava for CD31 and tdTomato (tdT, Sca1 + lineage). The right panel showed magnification of the boxed region. (B) Immunofluorescence staining of vein graft for CD31 and tdTomato. The right panel and the panel below showed magnification of the boxed regions. (C) Quantification of the percentage of CD31 + tdT + cells in CD31 + cells of both groups. (D) Sca1-CreER T2 ; Rosa26-tdTomato mice were bred with Rosa26-iDTR mice. Tamoxifen was given to label Sca1 + cells and diphtheria toxin (DT) injection was performed to ablate certain cells. Immunostaining for tdTomato and CD31 on vein graft sections after DT administration. For merge images, scale bars, 100 μm. For magnification images, scale bars, 20 μm. Data represent mean ± SEM, n = 5 in each group. p value was specified in the graph. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Injection, Immunostaining, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Isolated Sca1 + cells could differentiate into ECs with VEGF treatment in vitro (A) The morphological changes in Sca1 + cells after treatment with 50 ng/ml VEGF for 0–5 days. (B) mRNA expression level of EC markers Kdr , Flt1 , and Pecam1 as determined by RT-PCR at different time points after culture with VEGF. (C) Protein expression level of PECAM1, CDH5, and KDR as determined by western blotting assay. (D) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (E) Immunofluorescence staining of CD31 in Sca1 + cells treated with VEGF at different time points. Scale bars, 25 μm. Data represent mean ± SEM. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns p ≥ 0.05. n = 3 in each group. Statistical differences between groups were determined by one-way ANOVA analysis of variance with method of multiple comparisons.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Isolation, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: The effect of miR-145-5p on the differentiation of Sca1 + cells to ECs with VEGF treatment (A) RT-PCR analysis for the expression of miR-145-5p in Sca1 + cells treated with VEGF at different time points. (B) RT-PCR showed expression of miR-145-5p in Sca1 + cells after transfection with mimic control/miR-145-5p mimic and inhibitor control/miR-145-5p inhibitor. (C) and (D) RT-PCR analysis for the expression of Pecam1 , Flt1 , and Cdh5 in Sca1 + cells transfected with mimic control/miR-145 mimic or inhibitor control/miR-145 inhibitor and cultured in VEGF. (E) the protein expression level of PECAM1, CDH5 and KDR as determined by western blotting assay in each group. (F) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (G) Immunofluorescence staining of CD31 ( Pecam1 ) in Sca1 + cells and (H) analysis for fluorescence intensity of CD31 in each group, n = 3. scale bars, 25 μm. Data represent mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns p ≥ 0.05. Statistical differences between groups were determined by one-way analysis of variance with method of multiple comparisons. See also Figure S3 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, Cell Culture, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: The effects of miR-145-5p on the proliferation and migration of Sca1 + cells (A) CCK8 assay for the effect of miR-145-5p overexpression on the proliferation of Sca1 + cells. Transwell migration assay (B) and wound healing assay (C) for the effect of miR-145-5p overexpression on the migration of Sca1 + cells. n = 3 in each group. Scale bars, 25 μm. Data represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ns p ≥ 0.05. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Migration, CCK-8 Assay, Over Expression, Transwell Migration Assay, Wound Healing Assay, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: MiR-145-5p targets ERG to regulate differentiation of Sca1 + cells into ECs (A) The database TargetScan shows the combining site of miR-145-5p on ERG’s 3′-UTR region. (B) Luciferase activity of 293T cells co-transfected with wild type ERG-3′UTR and mimic control/miR-145-5p mimic or inhibitor control/miR-145-5p inhibitor. (C) Luciferase activity of Sca1 + cells co-transfected with mutant ERG-3′UTR and mimic control/miR-145-5p mimic or inhibitor control/miR-145-5p inhibitor. (D) Erg mRNA expression in Sca1 + cells transfected with mimic control or miR-145-5p mimic as measured by RT-PCR. (E) ERG expression of Sca1 + cells transfected with control plasmid vector (pcDNA-control) or plasmid to overexpress ERG (pcDNA-ERG) as measured by RT-PCR. (F) mRNA expression of Kdr , Pecam1 and Flt1 in Sca1 + cells transfected with pcDNA-control or pcDNA-ERG after treated with VEGF. (G) Protein expression level of KDR, PECAM1, and CDH5 in Sca1 + cells transfected with pcDNA-control or pcDNA-ERG after treated with VEGF. (H) mRNA expression of Kdr , Pecam1 and Flt1 in Sca1 + cells of each group as indicated. (I) Protein expression level of KDR, PECAM1, and CDH5 in Sca1 + cells of each group as indicated. (J) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (K) Expression level of miR-145-5p in healthy cephalic vein and arteriovenous fistula (AVF) as determined by RT-PCR. p value between the 2 groups were specified in the graph. n = 12 in the healthy group and n = 8 in the AVF group. Data represent mean ± SEM. Statistical differences between groups were determined by one-way ANOVA analysis of variance with method of multiple comparisons or (D,E,K) two-tailed Student’s t test for normally distributed values.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns p ≥ 0.05. See also Figure S4 - .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Transfection, Control, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet:

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Recombinant, Lysis, Purification, Luciferase, Reporter Assay, CCK-8 Assay, Plasmid Preparation, Software

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E efficiently suppresses human coronavirus spike-protein-mediated entry. (A) Levels of Ly6E, GILT, and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT, or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at 24 h after the addition of tet. The cellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). **, P < 0.001 compared to the control cells expressing CAT.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and lung cancer (A549) cells. (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of cellular LY6E expression was determined by Western blotting using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E-specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). Differences in viral RNA between scramble or LY6E-specific shRNA-expressing cells were analyzed statistically (**, P < 0.001; Student’s t test). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a Western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red); cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Identification of critical structural motifs essential for LY6E to restrict human coronavirus entry. (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is presented, in which the “three finger-fold” structure is highlighted with black boxes. The conserved L36 as well as the GPI anchor and N99 glycosylation sites are indicated. (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT or wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E. (D) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in medium with indicated concentrations of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (E) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E (L36A) were cultured with or without the indicated concentrations of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits TMPRSS2-enhanced entry of human coronaviruses. Flp-In T-Rex 293-derived cell lines expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. (A) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the absence of tet. (B) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the presence of tet. (C) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation ( n = 4); **, P < 0.001 compared to cells transfected with the pCAGGS vector.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Activity Assay, Standard Deviation

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Amphotericin B treatment compromises IFITM3 inhibition of human coronavirus entry, but have no impact on Ly6E inhibition of human coronavirus entry. Flp-In T-Rex 293-derived cell lines expressing IFITM3 (A) or LY6E (B) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1 μM AmphoB. Luciferase activity was measured at 48 h postinfection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mock treatment.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Inhibition, Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) Levels of Ly6E, GILT and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at the indicated time after the addition of tet. Intracellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). **, p <0.001 compared to the control cells expressing CAT.

Article Snippet: The codon-optimized (for human cells) SARS-CoV-2 spike gene, which is based on NCBI Reference Sequence YP_009724390.1, was purchased from GeneScript and cloned into pCAGGS vector as described previously ( ). pRS-derived retroviral vectors expressing a scramble shRNA and shRNA targeting the mRNA of human LY6E were obtained from OriGene (Cat. No. TR311641).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of intracellular LY6E expression was determined by Western blot using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations (n = 4). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red). Cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: The codon-optimized (for human cells) SARS-CoV-2 spike gene, which is based on NCBI Reference Sequence YP_009724390.1, was purchased from GeneScript and cloned into pCAGGS vector as described previously ( ). pRS-derived retroviral vectors expressing a scramble shRNA and shRNA targeting the mRNA of human LY6E were obtained from OriGene (Cat. No. TR311641).

Techniques: Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Infection, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is conducted and “three finger-fold” structure is highlighted with black box. The conserved L36 as well as GPI anchor and N99 glycosylation sites are indicated (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT, wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. Intracellular LY6E expression were detected by a Western blot assay. β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4).

Article Snippet: The codon-optimized (for human cells) SARS-CoV-2 spike gene, which is based on NCBI Reference Sequence YP_009724390.1, was purchased from GeneScript and cloned into pCAGGS vector as described previously ( ). pRS-derived retroviral vectors expressing a scramble shRNA and shRNA targeting the mRNA of human LY6E were obtained from OriGene (Cat. No. TR311641).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: Flp-In T-Rex 293-derived cell line expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48hpi. ( A ) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1). Error bars indicate the standard deviation (n=4). ( B ) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation (n=4). **, p <0.001, comparing to cells transfected with pCAGGS vector.

Article Snippet: The codon-optimized (for human cells) SARS-CoV-2 spike gene, which is based on NCBI Reference Sequence YP_009724390.1, was purchased from GeneScript and cloned into pCAGGS vector as described previously ( ). pRS-derived retroviral vectors expressing a scramble shRNA and shRNA targeting the mRNA of human LY6E were obtained from OriGene (Cat. No. TR311641).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Standard Deviation, Activity Assay

Journal: bioRxiv

Article Title: LY6E Restricts the Entry of Human Coronaviruses, including the currently pandemic SARS-CoV-2

doi: 10.1101/2020.04.02.021469

Figure Lengend Snippet: Flp-In T-Rex 293-derived cell line expressing IFITM3 ( A ) or LY6E ( B ) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1μM AmphoB. Luciferase activity was measured at 48 hr post-infection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations (n=4). **, p <0.001, compared to mock treatment.

Article Snippet: The codon-optimized (for human cells) SARS-CoV-2 spike gene, which is based on NCBI Reference Sequence YP_009724390.1, was purchased from GeneScript and cloned into pCAGGS vector as described previously ( ). pRS-derived retroviral vectors expressing a scramble shRNA and shRNA targeting the mRNA of human LY6E were obtained from OriGene (Cat. No. TR311641).

Techniques: Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay