Journal:

Article Title: Herpes Simplex Virus Type 2 Induces Rapid Cell Death and Functional Impairment of Murine Dendritic Cells In Vitro

doi: 10.1128/JVI.77.20.11139-11149.2003

Figure Lengend Snippet: Subpopulation of HSV-2-infected BMDC exhibit DNA breaks shortly after infection. BMDC were infected with wt HSV-2 at an MOI of 4 or left untreated, incubated for up to 12 h p.i., and then double stained for HSV ICP8 (anti-ICP8 MAb 383 plus FITC-conjugated goat anti-mouse MAb) and for apoptosis (biotin-labeled dCTP followed by avidin-Texas Red). Shown are immunofluorescence microscope images of the same optical section of a population of HSV-2-infected DC collected 2 h p.i. to demonstrate staining for ICP8 (green) (A), nick end-labeled DNA breaks (TUNEL) (red) (B), and a double-labeled, HSV-2-infected, apoptotic BMDC (yellow). Arrow in panels A, B, and C indicates positive staining of an apoptotic, HSV-2-infected BMDC. Included were double-stained uninfected BMDC as a negative control (D) and DNase-treated uninfected DC stained with biotin-dCTP-avidin-Texas red as a positive control (E) for the TUNEL assay. Magnification, ×40.

Article Snippet: Cytospin slides were prepared and fixed as described above, blocked with 20% goat serum (Jackson ImmunoResearch), and then stained with antibodies against HSV ICP4 (M612446; BioClone), ICP8 (383; a kind gift from David Knipe, Harvard Medical School, Boston, Mass.), or HSV-2 glycoprotein D (gD) (910; Biodesign International, Saco, Maine), followed by FITC-conjugated goat anti-mouse MAb (Jackson ImmunoResearch).

Techniques: Infection, Incubation, Staining, Labeling, Avidin-Biotin Assay, Immunofluorescence, Microscopy, TUNEL Assay, Negative Control, Positive Control

Journal: Frontiers in immunology

Article Title: NLRP3 promotes allergic responses to birch pollen extract in a model of intranasal sensitization.

doi: 10.3389/fimmu.2024.1393819

Figure Lengend Snippet: FIGURE 4 Allergic sensitization to BPE is independent of inflammasome-induced caspase 1 activation. (A) Immunofluorescence staining of lung sections from C57BL/6 and Nlrp3-/- mice (Figure 3) against ASC (red) and CD45 (white). Dapi (blue) was used to stain cell nuclei. Scale bar is 20 µm. The dashed line marks the epithelial cell barrier. b= bronchiole. (B) Quantification of ASC+/CD45+ cells of 3 mice/group, 1 image/mouse as depicted in (A). (C) BPE-specific serum IgE was measured via antigen-induced cross-linking of Fcϵ-receptors on RBL-2H3 cells and subsequent degranulation and release of b-hexosaminidase (b-hex release). (D, E) Cytokines secreted into BALF (D) or by restimulated splenocytes (E) were measured by Multiplex technology. The data are presented as box plots of at least 5 mice per treatment group of 3 individual experiments. Each mouse is depicted as one data point. One-way ANOVA with Tukey’s post hoc test was performed to determine statistical significance. *p<0.05, **p<0.01.

Article Snippet: The following primary antibodies were used: ASC/TMS1 (D2W8U) Rabbit mAb (1:200, RRID: AB_2799736, Cell Signaling Technology), Goat Anti-Mouse CD45 Polyclonal antibody (1:100, RRID: AB_442146, R&D Systems).

Techniques: Activation Assay, Staining, Multiplex Assay

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: Acta Pharmaceutica Sinica. B

Article Title: S -Allylmercaptocysteine improves alcoholic liver disease partly through a direct modulation of insulin receptor signaling

doi: 10.1016/j.apsb.2020.11.006

Figure Lengend Snippet: SAMC supplementation improved ALD-induced liver injury. (A) SAMC partially recovered reduced body weight of mice by ethanol administration from Day 6 to Day 16. (B) Changes of liver weight, liver-to-body weight ratio, and free fatty acid (FFA) of mice. (C) Changes of serum chemistry (ALT, AST, total cholesterol, and triglyceride) of mice. (D) NAS scorings and liver triglyceride (liver TG) results from each group of mice. (E) Changes of serum inflammatory mediators (TNF- α , and IL-6) of mice. (F) Representative Western blot results ( n = 3) and densitometry analysis of mice hepatic markers (G) Representative H&E, oil red O, and CD45 immunohistochemical staining images of the mice liver (yellow arrows indicate typical CD45 signals). Data are expressed as mean ± SEM ( n = 5). Significant differences between the indicated groups: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Scale bar = 20 μm.

Article Snippet: Primary antibodies against adiponectin (ADIPOQ; #ab22554), phosphorylated AKT at Thr308 or Ser473 (pAKT; #ab38449 and #ab81283), total AKT (#ab8805), aldehyde dehydrogenase 2 (ALDH2; #ab154993), adipose triglyceride lipase (ATGL; #ab109251), phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase at Ser487 (pAMPK; #ab131357), total AMPK (#ab32047), beta-actin ( β -actin; #ab115777), cleaved caspase-3 (C-caspase-3; #ab2302), carnitine palmitoyltransferase I (CPT1; #ab128568), cytochrome P450 2E1 (CYP2E1; #ab28146), CD45 (#ab205718), phosphorylated glycogen synthase kinase 3 beta at Ser9 or Tyr216 (pGSK3 β ; #ab75814 and #ab75745), total GSK3 β (#ab32391), nuclear factor erythroid 2-related factor 2 (NRF2; #ab156883), phosphorylated nuclear factor kappa B P65 subunit at Ser536 (pNF- κ B P65; #ab86299), total NF- κ B P65 (#ab16502), peroxisome proliferator-activated receptor alpha (PPAR α ; #ab24509), PPAR γ (#ab59256), and sterol regulatory element-binding protein 1c (SREBP-1c; #ab28481) were purchased from Abcam (Cambridge, UK).

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: Stem cell research & therapy

Article Title: Therapeutic role of uterine-derived stem cells in acute kidney injury.

doi: 10.1186/s13287-022-02789-0

Figure Lengend Snippet: Fig. 2 Labeled UDCs engraft the injured kidney as demonstrated by cell count, renal DsRed mRNA expression levels, in vivo imaging system and fluorescence-activated cell sorting (FACS). a Ds-Red positive uterine-derived cells (UDCs) were injected immediately following either sham surgery or bilateral renal ischemia–reperfusion injury. DsRed positive cells were preferentially incorporated into the injured kidneys as determined by FACS. b Similarly, elevated DsRed mRNA expression levels were higher in the kidneys of the renal ischemia–reperfusion injury group compared to the sham surgery group. c DsRed positive cell counts are shown in either male or female kidneys after ischemia–reperfusion injury. DsRed cells were preferentially incorporated in males. d After unilateral ischemia–reperfusion injured, more DsRed positive cells accumulated in the injured kidney compared to the non-injured kidney. e In vivo imaging of sham group and unilateral renal ischemia–reperfusion injury. DsRed positive uterine-derived cells (UDCs, shown here in green) were preferentially incorporated into the injured kidney. f and g demonstrate fluorescence-activated cell sorting of uninjured and ischemia–reperfusion injured kidneys. f DsRed labeled uterine-derived cells (UDCs) were not incorporated into the uninjured kidney in significant numbers. g Large numbers of DsRed cells were incorporated into the injured kidney. (N = 8 per group) *denotes statistical significance (p < 0.05) sham vs UDC; male vs female and non-injured vs injured

Article Snippet: Sections on slides were incubated at 4 °C overnight with primary antibodies, rabbit antiCD31 polyclonal antibody (catalog #ab28364 Abcam, Cambridge, MA, USA), goat anti-DsRed polyclonal antibody (catalog # sc-33354, Santa Cruz Biotechnology, Dallas, TX), and rat anti-CD45 antibody (catalog # ab23910, Abcam, Cambridge, MA) and anti-LTA monoclonal antibody (catalog # 10R-1174, Fitzgerald Industries International, North Acton, MA, USA).

Techniques: Labeling, Cell Counting, Expressing, In Vivo Imaging, Fluorescence, FACS, Derivative Assay, Injection

Journal: Stem cell research & therapy

Article Title: Therapeutic role of uterine-derived stem cells in acute kidney injury.

doi: 10.1186/s13287-022-02789-0

Figure Lengend Snippet: Fig. 3 Immunofluorescence staining of LTA, CD31, DAPI and DsRed in ischemia–reperfusion injured kidney. Colocalization of DsRed with either LTA or CD31 shows incorporation of endometrial stem cells into the renal tubules or vasculature, respectively. Scale bar: 50 μm

Article Snippet: Sections on slides were incubated at 4 °C overnight with primary antibodies, rabbit antiCD31 polyclonal antibody (catalog #ab28364 Abcam, Cambridge, MA, USA), goat anti-DsRed polyclonal antibody (catalog # sc-33354, Santa Cruz Biotechnology, Dallas, TX), and rat anti-CD45 antibody (catalog # ab23910, Abcam, Cambridge, MA) and anti-LTA monoclonal antibody (catalog # 10R-1174, Fitzgerald Industries International, North Acton, MA, USA).

Techniques: Immunofluorescence, Staining

Journal: Stem cell research & therapy

Article Title: Therapeutic role of uterine-derived stem cells in acute kidney injury.

doi: 10.1186/s13287-022-02789-0

Figure Lengend Snippet: Fig. 4 Serum creatinine levels and survival analysis. a Day 0 and b Day 2 serum creatinine levels in the DsRed positive uterine-derived cells (UDCs) treated group compared to controls, after renal ischemia–reperfusion injury. Each bar represents the mean ± SEM of two individual experiments and each experiment was performed in triplicate). *denotes statistical significance (p < 0.05) control vs UDC. c Survival analysis of control vs UDC treatment groups treatment groups; AKI indicates kidney with ischemia–reperfusion injury; Survival was improved after UDC treatment. *denotes statistical significance (p < 0.05) AKI + Hyst + CTL vs AKI + Hyst + UDC

Article Snippet: Sections on slides were incubated at 4 °C overnight with primary antibodies, rabbit antiCD31 polyclonal antibody (catalog #ab28364 Abcam, Cambridge, MA, USA), goat anti-DsRed polyclonal antibody (catalog # sc-33354, Santa Cruz Biotechnology, Dallas, TX), and rat anti-CD45 antibody (catalog # ab23910, Abcam, Cambridge, MA) and anti-LTA monoclonal antibody (catalog # 10R-1174, Fitzgerald Industries International, North Acton, MA, USA).

Techniques: Derivative Assay, Control

Journal: Stem cell research & therapy

Article Title: Therapeutic role of uterine-derived stem cells in acute kidney injury.

doi: 10.1186/s13287-022-02789-0

Figure Lengend Snippet: Fig. 5 Renal mRNA expression by qRT-PCR. Cxcl12, Bmp2, Bmp4 and Ctnf in the renal ischemia–reperfusion injury after treatment with DsRed positive uterine-derived cells (UDCs) vs control. N = 8 mice per group. Each bar represents the mean ± SEM of three individual experiments and each experiment was performed in duplicate). *denotes statistical significance (p < 0.05) control vs UDC

Article Snippet: Sections on slides were incubated at 4 °C overnight with primary antibodies, rabbit antiCD31 polyclonal antibody (catalog #ab28364 Abcam, Cambridge, MA, USA), goat anti-DsRed polyclonal antibody (catalog # sc-33354, Santa Cruz Biotechnology, Dallas, TX), and rat anti-CD45 antibody (catalog # ab23910, Abcam, Cambridge, MA) and anti-LTA monoclonal antibody (catalog # 10R-1174, Fitzgerald Industries International, North Acton, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Chemotaxis-driven disease-site targeting of therapeutic adult stem cells in dystrophic epidermolysis bullosa

doi: 10.1186/s13287-016-0388-y

Figure Lengend Snippet: Analysis of wild-type and DEB-affected skin infiltration with leukocytes. a H&E staining and indirect immunofluorescent analysis of skin infiltration with leukocytes. Detected antigens are shown in respective colors above the panels (CD45, green ; CXCR2, green ; CD11b, red ; colocalization of CD45 or CXCR2 and CD11b, yellow ). Nuclei were counterstained with DAPI ( blue ). bc blister cavity. Scale bar = 100 μm. b Quantitation of the skin-infiltrating cells. Data presented as an average number or percentage of cells (as indicated on the Y axis) per microscopic field ± SD. * p <0.05. ** p > 0.05. Data collected from at least 15 independent microscopic fields. CD-positive cell types are shown above the columns. H&E hematoxylin and eosin, WT wild-type skin, NB nonblistering, DEB dystrophic epidermolysis bullosa, Bl blistering DEB-affected skin

Article Snippet: For indirect immunofluorescent analysis, cross-sections were stained with the following primary antibodies: goat anti-mouse CD45 polyclonal (BD Bioscience), rat anti-mouse CD11b monoclonal (BD Bioscience), rabbit anti-CXCR2 polyclonal (Bioss, Woburn, MA, USA), mouse anti-FLuc monoclonal (Abcam, Cambridge, MA, USA), rabbit anti-type VII collagen polyclonal (Millipore, Billerica, MA, USA), and goat anti-type IV collagen polyclonal (Millipore).

Techniques: Staining, Quantitation Assay