Journal: Stem Cell Research & Therapy

Article Title: CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

doi: 10.1186/s13287-022-03026-4

Figure Lengend Snippet: Late passage (LP) MSC(AT) have a senescent transcriptome signature. A Gene set enrichment analysis demonstrates increased expression of genes known to be upregulated in senescence (shown in red) and reduced expression of genes downregulated in senescence (shown in blue) in LP-MSC(AT) and shows DPP4 among core enrichment genes; n = 1 pediatric and n = 1 adult, where EP = p6 and LP = p30. B – G Relative expression of selected transcripts from RNAseq analysis tested by quantitative RT-PCR over HPRT housekeeping gene in 9 additional samples. Expression of B CDKN2A (p16), C CDKN1A (p21), D DPP4 ( CD26), E HES1 , F SPP1 and G COL4A1 over the house-keeping gene HPRT . Data presented as mean ± SD, comparisons done with paired Wilcoxon test, * p < 0.05, ** p < 0.01

Article Snippet: The following primary antibodies were used at the dilutions indicated: CD26 (OriGene Technologies Inc., MD, USA; diluted 1:500), β-actin (Chemicon MAB1501; diluted 1: 100,000).

Techniques: Expressing, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

doi: 10.1186/s13287-022-03026-4

Figure Lengend Snippet: CD26 surface levels and total protein abundance in MSC(AT) increase with replicative senescence and age. A Western Blot of CD26 protein levels in MSC(AT) extracts from early passage (EP) and late passage (LP) pediatric ( n = 2) and adult ( n = 3) samples. Data represent mean + SEM, comparisons done with two-tailed Student’s t -test, * p < 0.05; ** p < 0.01. B Representative immunohistochemistry image of CD26 protein abundance in EP and LP-MSC(AT). C CD26 geometric mean fluorescence intensity (gMFI) and % CD26 high MSCs in EP and LP-MSC(AT) (6 adult and 4 pediatric MSC(AT), at EP = p4.1 ± 0.6 and LP = p21.0 ± 6.0). Data represent mean ± SD, comparisons done with paired Wilcoxon tests, ** p < 0.01. D CD26 gMFI and % CD26 high MSC(AT) in EP-MSC(AT) from pediatric and adult donors (6 pediatric MSC(AT) at p = 4.7 ± 0.5 and 6 adult MSC(AT) p = 3.8 ± 0.4). Data represent mean ± SD, comparisons done with unpaired Mann–Whitney tests, ** p < 0.01

Article Snippet: The following primary antibodies were used at the dilutions indicated: CD26 (OriGene Technologies Inc., MD, USA; diluted 1:500), β-actin (Chemicon MAB1501; diluted 1: 100,000).

Techniques: Western Blot, Two Tailed Test, Immunohistochemistry, Fluorescence, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

doi: 10.1186/s13287-022-03026-4

Figure Lengend Snippet: CD26 high MSC(AT) are less immunosuppressive than CD26 low MSC(AT). A Flow cytometry gating strategy for FACS separation of MSC(AT) based on CD26 surface abundance: CD26 low and CD26 high populations. B Immunopotency assay (i.e., MSC inhibition of proliferating CD4 + T cells). CD3CD28 activated T-cells were co-cultured with either CD26 low or CD26 high MSC(AT) at a 1:16 MSC:PBMC ratio. n = 4 adult and 1 pediatric MSC(AT). Data represent mean ± SD, comparisons done with paired Wilcoxon tests, * p < 0.05

Article Snippet: The following primary antibodies were used at the dilutions indicated: CD26 (OriGene Technologies Inc., MD, USA; diluted 1:500), β-actin (Chemicon MAB1501; diluted 1: 100,000).

Techniques: Flow Cytometry, Inhibition, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

doi: 10.1186/s13287-022-03026-4

Figure Lengend Snippet: Viability and CD26 surface levels in late passage MSC(AT) decrease following senolytic treatment. A Cell viability of late passage MSC(AT) that were either treated with DMSO (vehicle control) or a senolytic: NAVI: navitoclax (20 μM), QUER: quercetin (+:400 and ++:800 μM) and DMAG: 17-DMAG (+:25.6 and ++:51.2 μM). B CD26 gMFI and C %CD26 high MSCs among living MSC(AT). [ n = 4 adult and 1 pediatric LP-MSC(AT)]. Data presented as mean ± SD, comparisons done with paired one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The following primary antibodies were used at the dilutions indicated: CD26 (OriGene Technologies Inc., MD, USA; diluted 1:500), β-actin (Chemicon MAB1501; diluted 1: 100,000).

Techniques:

Journal:

Article Title: Co-Purification of Mac-2 Binding Protein with Galectin-3 and Association with Prostasomes in Human Semen

doi: 10.1002/pros.21287

Figure Lengend Snippet: Peptide Matches from Tandem Mass Spectrometry of Proteins Purified by Lactose Affinity Chromatography of Human Prostasomes

Article Snippet: Mouse monoclonal antibodies against human CD26 were from Lab Vision (Fremont, CA).

Techniques: Mass Spectrometry, Purification, Affinity Chromatography, Sequencing, Binding Assay

Journal:

Article Title: Co-Purification of Mac-2 Binding Protein with Galectin-3 and Association with Prostasomes in Human Semen

doi: 10.1002/pros.21287

Figure Lengend Snippet: Protein-protein interaction network of the identified proteins. Cytoscape was used to display the protein-protein interaction map generated from interaction database data pulled down from the HPRD, BioGRID, IntAct, and NCI/Nature Pathway Interaction databases. White nodes indicate the proteins identified in this study following β-galactoside affinity purification. Gray nodes indicate interacting proteins retrieved from the databases. Connecting lines indicate identified protein interactions. Each protein is shown by its gene symbol or entrez identification number. Galectin-3 (LGALS3), M2BP (LGALS3BP), lactoferrin (LTF), PIP (PIP), CD26 (DPP4), seminogelin I (SEMG1), seminogelin 2 (SEMG2), olfactomedin 4 (OLFM4).

Article Snippet: Mouse monoclonal antibodies against human CD26 were from Lab Vision (Fremont, CA).

Techniques: Generated, Affinity Purification

Journal:

Article Title: Co-Purification of Mac-2 Binding Protein with Galectin-3 and Association with Prostasomes in Human Semen

doi: 10.1002/pros.21287

Figure Lengend Snippet: Prostasome purification by size exclusion chromatography and M2BP immunoblot analysis. The membrane fraction from seminal plasma was subjected to size exclusion column chromatography on Sephacryl S300 and prostasomes were collected in the void volume (fractions 14-19). Electroblots of collected fractions were stained for total protein with Ponceau S and for M2BP (R&D polyclonal antibodies) and CD26 immunoreactivity. Molecular weight markers are indicated in kDa.

Article Snippet: Mouse monoclonal antibodies against human CD26 were from Lab Vision (Fremont, CA).

Techniques: Purification, Size-exclusion Chromatography, Western Blot, Column Chromatography, Staining, Molecular Weight

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Genome characteristics of pangolin MERS-like CoV, Manis javanica HKU4-related CoV (MjHKU4r-CoV) (A) Genome structure of MjHKU4r-CoV-1. (B) Similarity plot based on the full-length genome sequences. MjHKU4r-CoV-1 was used as a query sequence, Tylonycteris bat CoV HKU4-1, Pipistrellus bat CoV HKU5-1, bat MERS-related NeoCoV, and human MERS-CoV were used as reference sequences. (C) Phylogenetic tree based on the nucleotide sequences of the complete genomes of representative Alpha coronavirus (Alpha-CoV) and Beta coronavirus (Beta-CoV). MjHKU4r-CoV-1–4 are shown in red. Shaded colors represent different subgenera of coronaviruses. The scale bars represent 0.3 substitutions per nucleotide position. The software and setting used are described in the . (D) Schematic representation of the S protein. S1 and S2 subunits are indicated, as well as four domains within S1, including the N-terminal domain (NTD), RBD, subdomain 1 (SD1), and subdomain 2 (SD2). RBM alignment is shown at the bottom left, and the 16 key residues situated at the surface between MERS-CoV RBM and human DPP4 are pink shaded. Predicted furin cleavage sites of MjHKU4r-CoV-1 and the corresponding sites in other merbecoviruses are shown at the bottom right. The key arginine sites are shown in red, and the predicted furin cleavage sites are gray shaded. See also Figures S1 , , and and Table S1 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Sequencing, Software

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Isolation of MjHKU4r-CoV-1 and elucidation of its cell-entry mechanism (A) Transmission electron microscopy analysis of MjHKU4r-CoV-1 virions. Scale bars, 50 nm. (B) Cytopathic effect in Caco-2 cells at 48 h.p.i. with MjHKU4r-CoV-1 or mock infected (left, scale bars, 400 μm), and immunofluorescence assay (IFA) with an antibody against the Tylonycteris bat coronavirus HKU4 NP (right, scale bars, 200 μm). Red, NP; blue, nuclei. (C) Caco-2 cells were infected with MjHKU4r-CoV-1 at a multiplicity of infection (MOI) of 1 or 0.01. Viral titers and viral RNA copies at indicated time points were determined by TCID 50 assay and qRT-PCR, respectively. (D–G) Viral infection in Huh-7 cells with or without hDPP4 expression. Wild-type (WT) or DPP4 -knockout Huh-7 (KO) cells were infected with MjHKU4r-CoV-1 or MERS-CoV for 24 h. IFA was performed (D, scale bars, 200 μm) and quantified by high content screening (E), and viral titer (F) and RNA copy number (G) were determined in the supernatant. (H–K) Viral infection in KO cells after hDPP4 re-introduction. IF staining in Huh-7 KO cells transfected with hDPP4 expression plasmid or empty vector (H, scale bars, 200 μm) quantified by high content screening (I), and viral titer (J) and RNA copies (K) were determined in the supernatant at the indicated times. Data are presented as means and standard errors of the means (SEMs) of at least triplicate measurements in (E–G and I–K). Statistical significance was assessed using a two-tailed Student’s t -test in (E and I) and two-way ANOVA in (F, G, J, and K). See also Figure S4 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Infection, Immunofluorescence, Quantitative RT-PCR, Expressing, Knock-Out, High Content Screening, Staining, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 binds to human, bat, and pangolin DPP4 proteins (A) HeLa cells overexpressing the following DPP4 orthologous: hDPP4, Tylonycteris pachypus bat DPP4 ( Tp DPP4), Pipistrellus bat DPP4 ( Pp DPP4), or Manis javanica DPP4 ( Mj DPP4) were infected with MjHKU4r-CoV-1 at a MOI of 1 and subjected to IF staining at 24 h.p.i. Shown as MjHKU4r-CoV-1 NP or DPP4 protein expression (left, scale bars, 10 μm), or the replication dynamics of MjHKU4r-CoV-1 (viral titer, middle panel; genomic RNA, right panel). For IF staining, green, DPP4; red, NP; blue, nuclei. (B) Binding affinity of MjHKU4r-CoV-1, MERS-CoV, and HKU4-CoV RBD proteins to human, bat, and pangolin DPP4 proteins as measured by Bio-layer interferometry assay. The quantification of the binding affinity is shown in Table S2 . See also Figure S5 and Table S2 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Staining, Expressing, Binding Assay

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: Human cell tropism and human colon ex vivo infection of MjHKU4r-CoV-1, related to Figure 5 (A) NP staining (red) of the indicated human cell lines at 24 h and 72 h.p.i. with MjHKU4r-CoV-1. Blue, nuclei; scale bars, 200 μm. Replication of MjHKU4r-CoV-1 in cell culture supernatant was analyzed by TCID 50 assay. (B–D) qRT-PCR detection of the replication of MjHKU4r-CoV-1 in human ex vivo colon tissue (B) or culture supernatant (C). IF staining of human ex vivo colon at 24 h.p.i. with MjHKU4r-CoV-1 (D). Red, viral NP; green, human DPP4; blue, nuclei; scale bars, 100 μm.

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Ex Vivo, Infection, Staining, Cell Culture, Quantitative RT-PCR

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 infection in human colon organoids and airway organoids (A–C) Replication of MjHKU4r-CoV-1 in human colon organoids (hCO), shown as viral copy numbers in cells (A) and supernatants (B) and viral titers in supernatants (C) as analyzed by qRT-PCR and TCID 50 assay, respectively. (D) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human colon organoids at 72 h.p.i. (scale bars, 100 or 30 μm). Blue, nuclei. (E–G) Replication of MjHKU4r-CoV-1 in human airway organoids (hAWOs), shown as viral copy numbers in cells (E) and supernatants (F) and viral titers in supernatant (G) as analyzed by qRT-PCR and TCID 50 assay, respectively. (H) Co-staining of MjHKU4r-CoV-1 NP (red) and hDPP4 (green) in human airway organoids at 48 h.p.i. (scale bars, 50 or 10 μm). Blue, nuclei. See also Figure S6 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet: MjHKU4r-CoV-1 is infectious in hDPP4-Tg mice (A and B) hDPP4-Tg mice were intranasally inoculated with 1 × 10 6 TCID 50 MjHKU4r-CoV-1. (A) Mean body weights (n = 7 for MjHKU4r-CoV-1 infected mice and n = 3 for mock-infected mice), (B) body weights of individual mice. (C and D) qRT-PCR detection of viral replication in the lung (C) and brain (D) of MjHKU4r-CoV-1-infected hDPP4-Tg mice at the indicated time points (n = 4 for 1, 3, and 5 d.p.i. and n = 7 for 8 d.p.i.). (E) Lung and brain sections from MjHKU4r-CoV-1-infected or mock-infected mice stained for MjHKU4r-CoV-1 NP (red) at 5 d.p.i. (lung) and 8 d.p.i. (brain) (scale bars, 100 μm). Red, viral NP; blue, nuclei. (F) Pathological changes in the lungs of MjHKU4r-CoV-1-infected hDPP4-Tg mice at 5 d.p.i. Yellow arrows indicate severely affected areas (scale bars, 1,000 or 100 μm). See also Figure S7 .

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Infection, Quantitative RT-PCR, Staining

Journal: Cell

Article Title: A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry

doi: 10.1016/j.cell.2023.01.019

Figure Lengend Snippet:

Article Snippet: For the detection of hDPP4 protein in Huh-7 cells, the membrane was incubated with goat anti-hDPP4 polyclonal antibody (R&D, 0.3 μg/mL) and detected with horseradish peroxidase-conjugated rabbit anti-goat IgG (Proteintech, 1:5,000).

Techniques: Isolation, Recombinant, SYBR Green Assay, Luciferase, Reporter Assay, Sequencing, Knock-Out, Transgenic Assay, Plasmid Preparation, Software