Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: GB expresses CD73 in vitro and upregulates GB-CD73 in CD73−/− mice in vivo. A, B, Representative flow cytometry analysis of CD73 expression on GL261 (A) and U251 (B) GB cell lines. Data are representative of three experiments. C, Representative flow cytometry analysis of CD44 and CD73 expression on GL261. Data are representative of three experiments. D, G, Representative images of sham-implanted (D) and naive (G) mice brain sections stained with anti-CD73 antibody (brown) (n = 3). E, F, H, Quantification of CD73 expression in caudoputamen or cortex of sham-implanted or naive mice was performed by measuring positive pixel count using ImageScope analysis program (n = 3, three images analyzed/sample, Student's two-tailed t test). I, Representative images of brain sections from GL261-implanted mice stained with anti-CD73 antibody (brown) (n = 3–4). J, Quantification of CD73 expression inside tumors was performed by measuring positive pixel count using ImageScope analysis program (n = 3–4, 3 images analyzed/sample, Student's two-tailed t test). K, Densitometry quantification of CD73 protein expression inside tumors using ImageJ, normalizing to GAPDH and sham CTs (n = 4–5, Student's two-tailed t test). L, M, N, Flow cytometry analysis of CD73 expression percentage (L) (n = 3, Student's two-tailed t test), histogram (M), and median fluorescent intensity quantification (N) (n = 3, Student's two-tailed t test) on disassociated tumor from GB-bearing WT mice 3 weeks after implantation (n = 3, Student's two-tailed t test). Data are shown as mean ± SEM. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns, Nonsignificant.

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: In Vitro, In Vivo, Flow Cytometry, Expressing, Staining, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: Host CD73 promotes GB growth and invasion. A, C, Representative H&E-stained brain sections from GL261- or sham-implanted mice 3 weeks after implantation (A) (n = 6–7 per group) or 17 d after implantation (C) (n = 3 per group). Solid black lines are tumor borders. B, E, Quantification of H&E-stained brain sections (n = 6–7 per group, Student's two-tailed t test and one-way ANOVA, respectively). B, D, M, Tumor size is quantified by percentage of tumor area as follows: (tumor area/brain section area) × 100. E, I, N, Tumor invasiveness was scored based on invasion area, invasion distance, and meningeal invasion. (D, I, M, N) (n = 3 per group, Student's two-tailed t test for D, M, and N and one-way ANOVA for I). F, Schematic of CD73-FLK mice transgene construct. FW, Forward; RV, reverse. G, Representative images of naive CD73-FLK mice brain sections stained with anti-CD73 antibody (red), CD133 (green), and DAPI (blue) (n = 3). H, Representative images of sham-implanted WT and CD73-FLK mice brain sections stained with anti-CD73 antibody (brown) (n = 3). J, CD73-negative gating used for sorting GL261 cells. K, Flow cytometry analysis of CD73 expression on GL261CD73low cells. L, H&E-stained brain sections from GL261CD73low-implanted mice 3 weeks after implantation (n = 3). Solid black lines are tumor borders. O, GL261-implanted mice survival (n = 5–9 per group, Log-rank test). Data are shown as mean ± SEM. *p < 0.05, ns, Nonsignificant.

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: Staining, Two Tailed Test, Construct, Flow Cytometry, Expressing

Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: Absence of host CD73 inhibits GB angiogenesis and promotes glomeruloid vessel formation. A, Representative images of brain sections from GL261- or sham-implanted mice stained with anti-CD31 antibody (brown) (n = 4–6 per GB-implanted group and n = 2 for sham-implanted group). B, Quantification of vessel number per area (1.5 mm2) (n = 3–6 per GB-implanted group and n = 2 per sham-implanted group, three images analyzed/sample, one-way ANOVA). C, Vessel diameter measured by ImageScope software (n = 4–6 per group, three images analyzed/sample, one-way ANOVA). D, Quantification of glomeruloid vessel number per area (1.5 mm2) (n = 3–5 per group, 10 vessels analyzed/sample, one-way ANOVA). E, Representative images of brain sections from GL261-implanted mice 3 weeks after implantation stained with anti-CD31 (green), anti-Ki-67 antibody (red), and DAPI (blue). Arrowheads indicate Ki-67 endothelial nuclear expression and arrows indicate the absence of Ki-67 expression in endothelial nuclei (n = 3–4 per group). F, Ki-67 mean fluorescent intensity (MFI) quantified using Zen image analysis program (n = 10, 3 images analyzed/sample, 3 areas analyzed/image, Student's two-tailed t test). Data are shown as mean ± SEM. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001 (Student's two-tailed t test and one-way ANOVA).

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: Staining, Software, Expressing, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: Host CD73 promotes GB angiogenesis via regulating VEGF and α-dystroglycan. A, B, Representative images of brain sections from GL261- or sham-implanted mice stained with anti-CD31 (green), anti-VEGF antibody (red), and DAPI (blue) (n = 4–5 per GB-implanted group and n = 2 per sham-implanted group). C, VEGF mean fluorescent intensity (MFI) quantified using Zen image analysis program (n = 4–5 per GB-implanted group and n = 2 per sham-implanted group, 2–6 images analyzed/sample, one-way ANOVA). D, Western blot densitometry analysis of VEGF (n = 5–7 per group, one-way ANOVA). E, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-CD31 (green), anti-α-dystroglycan antibody (red), and DAPI (blue); images taken from inside the tumor (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group). F, α-dystroglycan MFI inside the tumor quantified using Zen image analysis program (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group, five areas analyzed/sample, one-way ANOVA). Data are shown as mean ± SEM. *p < 0.05, ****p < 0.0001.

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: CD73 regulates MMP2, MMP9, and TIMP1 to promote GB invasiveness and angiogenesis. A, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-CD31 (green), anti-MMP2 antibody (red), and DAPI (blue) (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group). B, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-MMP2 antibody (brown) (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group). C, MMP2 immunohistochemistry staining positive pixel count was quantified using ImageScope analysis program (n = 3–4 per group, three images analyzed/sample, one-way ANOVA). D, qRT-PCR analysis of TIMP2 mRNA collected from GL261- or sham-implanted mice (3 weeks after implantation) (n = 8–11 per GB-implanted group and n = 2–3 per sham-implanted group, one-way ANOVA). E, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-MMP9 antibody (green) and DAPI (blue); images taken from the edge of the tumor (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group). F, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-MMP9 antibody (brown) (n = 3–4 per GB-implanted group and n = 2 per sham-implanted group). G, Representative images of brain sections from GL261- or sham-implanted mice (3 weeks after implantation) stained with anti-TIMP1 (green), anti-MMP9 antibody (red), and DAPI (blue); images taken from the edge of the tumor (n = 4–5 per GB-implanted group and n = 2 per sham-implanted group). H, MMP9 immunohistochemistry staining positive pixel count on the tumor edge quantified using ImageScope analysis program (n = 3–4 per group, three images analyzed/sample, one-way ANOVA). I, TIMP1 MFI in brain sections quantified using Zen image analysis program (n = 4–5, three images analyzed/sample, one-way ANOVA). J, MMP activity of brain tissue homogenates from GB-bearing mice (3 weeks after implantation) normalized to sham-implanted CTs (n = 4–7, Student's two-tailed t test). Data are shown as mean ± SEM. *p < 0.05, **p ≤ 0.01, ****p < 0.0001.

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: Staining, Immunohistochemistry, Quantitative RT-PCR, Activity Assay, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A 2B Adenosine Receptor Signaling

doi: 10.1523/JNEUROSCI.1118-18.2019

Figure Lengend Snippet: Working model describing how CD73 promotes GB pathogenesis. A, Host CD73 promotes GB invasiveness by upregulating MMP9 and inhibiting its inhibitor, TIMP1. B, Host CD73 upregulates angiogenesis by promoting mother vessels to divide into smaller vessels, thereby increasing the vessel density in GB (solid arrows). In the absence of host CD73, GB upregulates GB-CD73 and increases MMP2 expression through A2B AR signaling. GB-CD73 promotes VEGF and α-dystroglycan expression, which leads to intravascular endothelial cell proliferation and glomeruloid vessel formation in GB (dotted arrows). C, CD73-generated adenosine acts via the A2B AR to promote P-gp and MRP1 upregulation, which results in increased drug efflux, thereby increasing GB chemoresistance.

Article Snippet: Membrane was incubated with antibodies against A 1 AR, A 2A AR, A 2B AR, A 3 AR, P-gp, CD73, and VEGFA (1:2000; Alomone Labs, AAR-006, AAR-002, AAR-003, AAR-004; 1:1000, GeneTex, GTX108354; 1:2000, Abgent, AP2014b; 1:1000; Abcam, ab46154, respectively) overnight at 4°C.

Techniques: Expressing, Generated

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet:

Article Snippet: Anti-5′-Nucleotidase Clone 4G6E3 , Novus Bio , Cat# NBP2-37271.

Techniques: Recombinant, Blocking Assay, Western Blot, Cell Isolation, RNA Sequencing Assay, CRISPR, Plasmid Preparation, Software

Journal: Journal of Tissue Engineering

Article Title: Therapeutic potential of epiphyseal growth plate cells for bone regeneration in an osteoporosis model

doi: 10.1177/20417314221116754

Figure Lengend Snippet: Cellular characterization of EGPCs and BM-MSCs: (a) observation of cell morphology and measurement of cell shape (length to width ratio), (b) the proliferation rates of EGPCs and BM-MSCs were measured from day 1 to day 7 by the CCK-8, (c) cell-cycle analysis of BM-MSCs and EGPCs by using propidium-iodide staining. Quantitation of the cells at each-cycle phase, and (d) Flow-cytometric analyses for the surface markers on BM-MSCs and EGPCs. Representative flow-cytometry profiles are depicted for CD73, CD44, CD90, and CD200. Black and red plots represent the negative control and experimental samples, respectively.

Article Snippet: Antibodies against CD73 (NBP2-25235; 5′-Nucleotidase/CD73 Antibody, NOVUS BIOLOGICALS), CD44 (MCA643GA; MOUSE ANTI RAT CD44, Bio-Rad, UK), CD90 (sc-53456; Thy-1/CD90 antibody (aTHy-1A1), Santa Cruz Biotechnology), and CD200 (MA1-70035; CD200 Monoclonal Antibody (OX-2), ThermoFisher Scientific) were used as the primary antibodies (1:100 diluted in the FACS buffer).

Techniques: CCK-8 Assay, Cell Cycle Assay, Staining, Quantitation Assay, Flow Cytometry, Negative Control