Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

doi: 10.1165/rcmb.2015-0095oc

Figure Lengend Snippet: Figure 1: PDGFRα+GFP mice reveal two distinct fibroblast phenotypes (A) Single cells isolated from adult mouse lungs were sorted into Lin-: (CD45-, CD31-, CD326-) and

Article Snippet: MACS (miltenyl Biotec) anti-mouse CD45 MicroBeads (for negative selection), Anti-Mouse CD140α Biotin (PDGFRα) (miltenyl Biotec), Anti-Mouse CD326 Biotin (EpCAM)(ebioscience), and AntiBiotin MicroBeads (MASC, Cat# 130-090-485).

Techniques: Isolation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

doi: 10.1165/rcmb.2015-0095oc

Figure Lengend Snippet: Figure 2: Immunophenotyping of PDGFRα+ and PDGFRα- fibroblasts in adult mouse lungs. Lin- (CD45-CD326-CD31-) stromal cells were gated for CD140α+ (green) or CD140α- (red) subpopulations and expression of SCA-1 (A), CD29 (integrin β1) (B), CD34 (C), Thy1(CD90) (D), CD44, CD49 and CD40 (E) compared between the CD140α+ and CD140α- fibroblasts. Histograms and bivariate contour plots of surface

Article Snippet: MACS (miltenyl Biotec) anti-mouse CD45 MicroBeads (for negative selection), Anti-Mouse CD140α Biotin (PDGFRα) (miltenyl Biotec), Anti-Mouse CD326 Biotin (EpCAM)(ebioscience), and AntiBiotin MicroBeads (MASC, Cat# 130-090-485).

Techniques: Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: Phenotype and immunostimulatory activity of renal mononuclear phagocytes. (A) Representative contour plot for CD11c (APC-Cy7) and F4/80 (APC) expression on pregated CD45+ renal single cells. (B) Representative contour plots for IRF4 (FITC) and IRF8 (PE) staining on pregated CD11c−F4/80+CD45+, CD11c+F4/80+CD45+, and CD11c+F4/80−CD45+ renal single cells. (C) Frequency of IFN-γ+ and IL-17+ alloreactive Balb/c T cells by ELISpot after 2 days of exposure to CD11c−F4/80+, CD11c+F4/80+, or CD11c+F4/80− cells isolated from C57/BL6 kidneys. Mean±SD of three replicates from one representative experiment, *P<0.05. (D) Frequency of IFN-γ+ and IL-17+ alloreactive Balb/c T cells by ELISpot after 2 days of exposure to renal CD11c+F4/80+ cells either unstimulated or LPS-stimulated; *P<0.05. (E and F) Frequency of IFN-γ+ (E) and IL-17+ (F) OVA-specific OT2-CD4+ (TH1 or TH17) or OT1-CD8+ (TC1 or TC17) T cell clones detected by ELISpot after 2 days of exposure to renal CD11c+F4/80+ cells (OVA-transgenic or wild-type). Wild-type renal CD11c+F4/80+ cells were exposed or not to OVA, either in vitro (100 μg/ml) during MLR or in vivo by intravenous infusion (1mg/500 μl per mouse). OVA-exposed (both in vitro and in vivo) renal CD11c+F4/80+ cells were either stimulated with LPS or left unstimulated during the MLR. Values are mean±SD, n=3–4 independent experiments for each condition; *P<0.05. ND, not detectable.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Activity Assay, Expressing, Staining, Enzyme-linked Immunospot, Isolation, Clone Assay, Transgenic Assay, In Vitro, In Vivo

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: CI injury modifies the phenotype and the immunostimulatory activity of renal CD11c+F4/80+ MΦs. (A) Cell counts of CD11c+F4/80+CD45+, CD11c+F4/80−CD45+, and CD11c−F4/80+CD45+ renal single cells from kidneys subjected or not to 16 hours of CI. Mean±SD, n=4 independent experiments; *P<0.05. (B) Log-fold change of median fluorescence intensity (MFI) of different markers on CD11c+F4/80+ rMΦs from kidneys subjected to CI compared with nonischemic kidneys. Mean±SD, n=3–4 independent experiments for each molecule; *P<0.05 versus pre-CI MFI. (C) T cell proliferation (3H-thymidine incorporation) of alloreactive Balb/c T cells after 2 days of exposure to CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours of CI. Values are the three replicates of a representative experiment; *P<0.05. (D) Representative images and results summary of IFN-γ+ and IL-17+ alloreactive Balb/c T cell frequency assessed by ELISpot after 2 days of exposure to renal CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours CI and used either unstimulated or LPS-stimulated. Mean±SD, n=7 independent experiments for IFN-γ+ and n=4 independent experiments for IL-17+; *P<0.05. (E and F) Frequency of IFN-γ+ (E) and IL-17+ (F) OVA-specific OT2-CD4+ or OT1-CD8+ T cells by ELISpot after 2 days of exposure to CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours CI. Renal CD11c+F4/80+ rMΦs were exposed to OVA in vivo by intravenous infusion and used either unstimulated or LPS-stimulated in vitro during MLR. Mean±SD, n=3–5 independent experiments for each condition; *P<0.05.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Activity Assay, Fluorescence, Isolation, Enzyme-linked Immunospot, In Vivo, In Vitro

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: Donor CD11c+F4/80+ rMΦs cell count and phenotype in transplantation-induced I/RI. (A and B) Cell counts of total donor cells (CD45.2+), donor rMΦs (CD45.2+CD11c+F4/80+), total recipient cells (CD45.1+), and recipient rMΦs (CD45.1+CD11c+F4/80+) in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts pretransplant (pre-tx) and at days 1, 3–4, and 7–10 post-transplant. Mean±SD, n=3–7 mice at each time point; *P<0.05 versus pre-tx, ND: not detectable. (C) Percentage of proliferating (Ki67+) cells on donor rMΦs in wild-type kidney grafts pretransplant and at days 1–3 and 7–10 post-transplant. Mean±SD, n=2–3 mice at each time point; *P<0.05 versus pre-tx. (D and E) Post-transplant log-fold change in median fluorescence intensity (MFI) of selected molecules in donor rMΦs compared with pretransplant values, both in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at days 1–3 (D) and 3–7 (E) post-transplantation. Mean±SD, n=2–6 mice for each molecule at each time point; *P<0.05. (F) iNOS, TNFα, and Ym1 mRNA expression in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at day 7 post-transplant. Mean±SD, n=3–4 mice; *P<0.05. (G) Semiquantitative scores (0–3) and representative images of nitro-tyrosine staining in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at day 7 post-transplant. *P<0.05. Original magnification, ×400.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Cell Counting, Transplantation Assay, Fluorescence, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Jianpi Yangzheng Xiaozheng decoction alleviates gastric cancer progression via suppressing exosomal PD-L1

doi: 10.3389/fphar.2023.1159829

Figure Lengend Snippet: JPYZXZ reduced the number of MDSCs in tumor bearing mice. (A,B) Percentage of MDSCs (CD45 + CD11b+Gr1+) in blood determined using flow cytometry. (C,D) Percentage of MDSCs (CD45 + CD11b+Gr1+) in tumors determined using flow cytometry. (E) Immunofluorescence staining for DAPI (nucleus), CD11b, Gr-1, and PD-L1 in tumor tissue sections from the xenograft tumor model. Data are represented as the mean ± SEM, n = 5 mice or n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The single-cell suspensions (1 × 10 7 cells/mL) were stained with fluorochrome-coupled antibodies AF488-CD45 (Elabscience, E-AB-F1136L), PE/Dazzle™594-CD11b (BioLegend, 101256), and APC-Gr-1 (BioLegend, 108412) for 30 min at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 4. A, expression of the ST6Gal I in the T200 cell line, which does not express CD45, did not result in increased SNA binding. Nine clones expressing ST6Gal I mRNA were examined, but none demon- strated increased SNA binding by flow cytometry; clone SNA.T1 is shown for example. C.T1 is one of nine control clones transfected with vector alone. B, SNA blotting of whole cell extracts of C.T1 and SNA.T1 cells did not demonstrate any differences in staining between the two clones. C, T200 clones express ST6Gal I mRNA and protein. RT-PCR and immunoblot analysis of nine clones demonstrated ST6Gal I expres- sion, as shown for the SNA.T1 clone, with no ST6Gal I expression in any of the controls, as shown for the C.T1 clone. The samples are represent- ative of all 18 clones examined. The expressed protein is enzymatically active, as demonstrated by the addition of sialic acid to asialofetuin. Asialofetuin was incubated with lysates of C.T1 or SNA.T1 cells and precipitated with anti-fetuin, and 2,6-linked sialic acid was detected by SNA blotting. Weak SNA reactivity of fetuin incubated with extract of C.T1 cells may reflect the addition of 2,6-linked sialic acid to O-glycans, because no SNA reactivity was detected with the asialofe- tuin acceptor substrate alone (not shown). Densitometric analysis of the SNA-binding bands was performed; the ratio of SNA binding to fetuin incubated with SNA.T1 extract compared with C.T1 extract was 6.3:1.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Clone Assay, Flow Cytometry, Control, Transfection, Plasmid Preparation, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 3. The ST6Gal I preferentially sialylates N-glycans on CD45. A, total SNA-binding glycoproteins were precipitated from con- trol clones transfected with vector alone (lanes C.2 and C.4) or from the SNA.1 clone expressing the ST6Gal I. Precipitated glycoproteins were probed with biotinylated SNA. The only significant difference in the profile of SNA binding glycoproteins was an increase in a band with a mass of 200 kDa (arrow). B, the SNA reactive band is CD45. The cells were cultured in 2 mM DMNJ, as above, or in medium alone. The cell lysates were precipitated with CD45 antibody or SNA (indicated below) and probed with CD45 antibody. The band with increased SNA staining reacts with both SNA and antibody to CD45. In addition, the increased SNA binding to CD45 is abolished by pretreatment with DMNJ, which blocks synthesis of complex N-glycans. In both blots, the width of the CD45 band is diminished in DMNJ-treated cells compared with cells expressing the ST6Gal I, as a result of decreased complexity of glyco- sylation. C, increased SNA binding to CD45 results from sialylation of N-glycans. CD45 was detected in whole cell lysates of SNA.9 cells by immunoblotting (top panel). The CD45 bands were excised and incu- bated with or without PNGase F, as indicated, and reprobed with SNA-biotin. Removal of N-glycans from CD45 by PNGase F treatment reduced SNA binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Clone Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 5. ST6Gal I expression inhibits galectin-1-induced segre- gation of CD45. C.4 or SNA.9 cells were treated with galectin-1 or buffer control and fixed, and cell surface CD45 was detected by immu- nofluorescence. A, cells were analyzed by confocal microscopy to detect CD45 segregation. B, the percentage of cells demonstrating segregation of CD45 was scored by counting 50 cells in six fields. The cells were treated with buffer control (open bars) or galectin-1 (shaded bars). The SNA.9 cells demonstrated a marked reduction in CD45 segregation, compared with CD45 segregation in control C.4 cells. The percentage of cell death for a parallel sample is indicated by the numbers above each bar.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Control, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 6. ST6Gal I expression inhibits galectin-1-induced modu- lation of CD45 protein-tyrosine phosphatase activity. A, CD45 is the major PTP in PhaR2.1 cells. Whole cell lysates of the CD45 paren- tal cell line, PhaR2.1, and the CD45 derivative T200, were assayed for PTP activity in the presence (solid bar) or absence (open bar) of the PTP inhibitor bp V (phen). PTP activity was measured by the release of p-nitrophenol, detected at 415 nm. B, ST6Gal I expression abrogates the decrease in PTP activity triggered by binding of galectin-1. C.2 cells (open symbols) and SNA.9 cells (closed symbols) were incubated with 30 g of galectin-1 for the indicated times at 37 °C. At the indicated times, the cells were lysed, and PTP activity in whole cell lysates was meas- ured as described under “Experimental Procedures,” in the presence (squares) or absence (circles) of the PTP inhibitor bpV (phen). C.2 cells demonstrate a 40% reduction in PTP activity 1 min after galectin-1 binding. SNA.9 cells demonstrate no change in PTP activity after ga- lectin-1 binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation

Journal: Journal of Virology

Article Title: Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

doi: 10.1128/jvi.00189-10

Figure Lengend Snippet: FIG. 2. Specific interaction of UL112-113 p84 with UL44 among the six replication core proteins. (A) X-Gal filter assays of yeast cells expressing both the GAL4-DB/UL112-113 (p34, p43, p50, and p84) fusion proteins and the GAL4-A/replication core (UL44, UL54, UL57, UL70, UL102, and UL105) fusion proteins. The cells expressing the GAL4-A proteins alone were used as a control. Green indicates a positive interaction. (B) 293T cells were cotransfected with plasmids encoding HA-tagged UL112-113 proteins and Myc-tagged replication core proteins (UL44, UL54, UL57, UL70, UL102, or UL105). (Top) At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting with anti-HA Ab. (Middle and bottom) Total cell lysates were also immunoblotted with anti-Myc or anti-HA Abs. (C) HF cells were infected with HCMV Towne at an MOI of 2.0. Total cell lysates were prepared 2 days postinfection and immunoprecipitated with M23 Ab specific for the UL112-113 proteins, followed by immunoblotting with anti-PCNA or anti-p84 Abs. Total cell lysates were also immunoblotted with anti-p84 or anti-PCNA Abs to show the protein expression levels.

Article Snippet: Anti-proliferating cell nuclear antigen (PCNA) mouse MAb PC10 was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Control, Immunoprecipitation, Western Blot, Infection