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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: Sca1+ microbeads kit , Miltenyi Biotec , Cat#130-122-615.

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: iScience

Article Title: Deciphering postnatal limb development at single-cell resolution

doi: 10.1016/j.isci.2022.105808

Figure Lengend Snippet: Interpretation of Cd34 + cell cluster and Ly6e + cell cluster in developmental articular cartilage (A) Identities of cells from AC (corresponding to Figure 2 A) by UMAP visualization revealing four cell types including typical AC, chondrocytes interconnected with articular cartilage and enthesis (AC_EN), chondrocytes interconnected with articular cartilage and growth plate (AC_GP), and cells in cell cycle from the above three subsets (AC_ Mki67 + ). (B) Fraction of cells from different postnatal stages in each subset in (A). (C) Identities of cells from typical AC (corresponding to A) by UMAP visualization revealing four cell subsets referred to AC1 to AC4. (D) Developmental trajectory inferred by RNA velocity and visualized on the UMAP projection (corresponding to C). (E) Dot plots showing the expression of curated feature genes of each subset in (C) (Left) and the UMAP plots showing the expression of representative genes encoding cell membrane proteins ( Cd34 in AC2 subset and Ly6e in AC4 subset). (F) UMAP plots showing the distribution of Cd34 + cell subset, Ly6e + cell subset, and Cd34 - Ly6e − cell subset. Noting that there were no cells co-expressed Cd34 and Ly6e . (G) Line chart showing the temporal variation of fraction of each subset at different postnatal stages. (H) Expression heatmap of curated TFs of Cd34 + cell subset and Ly6e + cell subset following pseudotime analysis. (I) Dot plots showing the expression of feature genes denoting stem or progenitor character in Cd34 + cell subset, Ly6e + cell subset, and Cd34 - Ly6e − cell subset. (J) Immunofluorescence staining showing Ly6e distribution in murine hind limbs at different stages, including E14.5, E16.5, P2, P5, P14, and M2 (n = 6 at E14.5, n = 5 at E16.5, and n = 4 at other time points). (K) Immunofluorescence staining showing Cd34 distribution in murine hind limbs at different stages, including E14.5, E16.5, P2, P4, P8, P14, P28, and M2 (n = 6 at E14.5, n = 5 at E16.5, and n = 4 at other time points). E: embryonic, P: postnatal. Scale bars, 50 μm. Blue indicates DAPI staining of nuclei and red indicates Ly6e or Cd34 staining. I, Interzone; S, synovium; T, tibia; M, meniscus.

Article Snippet: Ly6e antibody , Biorbyt , Cat# orb20596; RRID: AB_10751664.

Techniques: Expressing, Membrane, Immunofluorescence, Staining

Journal: iScience

Article Title: Deciphering postnatal limb development at single-cell resolution

doi: 10.1016/j.isci.2022.105808

Figure Lengend Snippet: Delineation of Cd34 + cell cluster and Ly6e + cell cluster in enthesis cells (A) UMAP plots showing expression patterns of Cd34 and Ly6e in all delineated cells and fraction of the positive expressed cells in each cell type. (B) UMAP visualization of Cd34 + cells screened from AC, GP, and enthesis corresponding to Figure 2 A revealing four subsets (Left) and fraction of cells from different cell types in each subset (Right). (C) Dot plots showing the expression of curated feature genes marked for GP, articular cartilage (AC), enthesis (EN), and cells in cell cycle (PRO) in each subset in (B) (Right). (D) UMAP visualization of Ly6e + cells screened from AC, GP, and enthesis corresponding to Figure 2 A revealing three subsets (Left) and fraction of cells from different cell types in each subset (Right). (E) Dot plots showing the expression of curated feature genes marked for GP, articular cartilage (AC), enthesis (EN), and cells in cell cycle (PRO) in each subset in (B) (Right). (F) UMAP visualization of enthesis cells revealing three subsets. (G) Dot plots showing feature genes expression of each subset in enthesis. (H) Pseudotime trajectory analysis of all subsets in enthesis predicted two major developmental branches with arrows indicating the predicted direction. (I) Distribution of cells expressing stem/progenitor marker genes ( Tppp3 and Cd55 ), terminal differentiation marker gene ( Alpl ), Cd34 , Ly6e , and Ly6a along the pseudotime axis. (J) UMAP plots showing the distribution pattern of Cd34 + subset, Ly6e + subset, Cd34 + Ly6e + subset, and Cd34 − Ly6e − subset in enthesis and the cell fraction distribution in different subset corresponding to F. (K) Pseudotime trajectory showing distribution of Cd34 + subset, Ly6e + subset, Cd34 + Ly6e + subset, and Cd34 − Ly6e − subset. (L) Expression heatmap of curated TFs following pseudotime analysis displaying five clustering patterns with arrows indicating the predicted developmental direction corresponding to (H).

Article Snippet: Ly6e antibody , Biorbyt , Cat# orb20596; RRID: AB_10751664.

Techniques: Expressing, Marker

Journal: iScience

Article Title: Deciphering postnatal limb development at single-cell resolution

doi: 10.1016/j.isci.2022.105808

Figure Lengend Snippet:

Article Snippet: Ly6e antibody , Biorbyt , Cat# orb20596; RRID: AB_10751664.

Techniques: Recombinant, Software

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for molecular cloning

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for qRT-PCR

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: NAT10 targets LY6E, which mediates ac4C modifications during SINV infection. ( A ) Validation of candidate genes from the RNAseq data using qRT-PCR in NAT10-KD cells infected with SINV. ( B ) Predicted ac4C modification sites for the LY6E pre-mRNA and mature mRNA. ( C ) IP of 293T cells transfected with the NAT10-Myc plasmid and anti-Myc antibody; enriched LY6E mRNA was analyzed using qRT-PCR; the interactions between the NAT10 and LY6E mRNA were also analyzed. ( D ) ( Upper panel ) Immunoblot of the NAT10 immunoprecipitate in panel C. ( Lower panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR in panel C. ( E ) After incubating with the anti-ac4C antibody and normal rabbit IgG mixed with protein A/G beads at 4°C for 2 h, respectively, incubation was continued with the NAT10-KD Huh7 cell lysate for 2 h. The bound ac4C-modified RNA was eluted and analyzed using qRT-PCR. ( Left panel ) The ac4C-modified RNA was also analyzed using qRT-PCR. ( Right panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR. Equal amounts of RNA fragments not subjected to immunoprecipitation were used as the input controls. ( F ) ( Upper panel ) Schematic of the 4xS1m aptamer. ( Lower panel ) WT or ac4C site mutated (C–T mut) LY6E mRNA tagged with 4xS1m aptamer was incubated with cell lysates overexpressing NAT10 and separated via streptavidin-conjugated beads. NAT10 in the cell lysate was pulled down, and the LY6E mRNA was detected using an immunoblot. Cells transfected with vectors were used as negative controls. ( G ) ( Upper panel ) Schematic diagram of the dual-luciferase reporter plasmid pmirGLO. ( Lower panel ) Luciferase activity in the NAT10-KD Huh7 ( I ) or A549 (ii) cells transfected with pmirGLO with the WT or ac4C-modifier-site-mutated (C–T mut) the 3′-UTR of the LY6E mRNA. Firefly luciferase activity was normalized to Renilla luciferase activity. ( H, I ) Stability of LY6E mRNA in NAT10-KD Huh7 ( H ) and A549 ( I ) cells after treatment with actinomycin D (5 µg/mL) was analyzed using qRT-PCR at different time points. ( J ) Ly6E mRNA levels were analyzed in Huh7 cells using qRT-PCR at different time points after 24 h of Remodelin treatment with actinomycin D. Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and NS, not significant (A, E, G, H, I, and J, two-way ANOVA with Bonferroni post-test; C, unpaired Student’s t -tests).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Biomarker Discovery, Quantitative RT-PCR, Modification, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, Amplification, Incubation, Immunoprecipitation, Luciferase, Activity Assay, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Predicted ac4C modification sites in the LY6E mRNA determined using PACES ^a

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Mutations in the predicted ac4C modification sites of the LY6E mRNA ^a , ^b

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: SINV is positively affected by NAT10 as it regulates the stability of the LY6E mRNA. ( A ) qRT-PCR analysis of LY6E mRNA expression in LY6E-KD Huh7 cells. ( B ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells at 24 hpi (MOI = 1). ( C ) Immunoblot analysis of the SINV capsid protein expression in LY6E-KD Huh7 cells at 6, 12, and 24 hpi (MOI = 1). ( D ) Plaque formation assay using the SINV infectious virions obtained from the LY6E-KD Huh7 cell culture medium at 24 hpi (MOI = 1). ( E ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( F ) Immunoblot analysis of the SINV capsid protein abundance described in panel ( E ). ( G ) Plaque formation assay using the SINV infectious virions obtained from the culture supernatant described in panel ( E ). ( H ) qRT-PCR analysis of the SINV RNA expression levels in NAT10-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( I ) Immunoblot analysis of the SINV capsid protein abundance as described in panel ( H ). ( J ) Plaque formation assay for the SINV infectious virions obtained from the culture supernatant described in panel ( H ). Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05 and *** P ≤ 0.001 (A, B, and D, unpaired Student’s t -tests; E, G, H, and J, one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Western Blot, Plaque Formation Assay, Cell Culture, Infection, Quantitative Proteomics, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Working model showing how the loss of NAT10 reduces alphavirus replication. Alphavirus (SINV) infection upregulates NAT10 in host cells and promotes NAT10-mediated ac4C acetylation of LY6E mRNA transcripts, increasing LY6E expression and enhancing alphavirus replication.

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Expressing