Journal: Molecular neurobiology

Article Title: 3'poly-G-tailed ODNs inhibit F-spondin to induce cell death and neurite retraction in rat embryonic neurons.

doi: 10.1007/s12035-012-8275-8

Figure Lengend Snippet: Fig. 1 CpG-A ODNs induced cell aggregation and neurite retraction of embryonic neuron cells TLR9-independently. a Embryonic hippocampal neu- rons (EHN) were isolated and purified from E18 SD rat em- bryo. TLR9 protein expression in RAW264.7 cells (RAW), EHN, and human embryonic kidney 293 cells (HEK) was an- alyzed by western blotting. Actin was used as a loading control. b After culture of rat embryonic neuron cells for 2 days in vitro, 1 μM CpG-B ODN (ODN 2006) or CpG-A ODN (ODN 2216) was added directly into the cul- ture medium. After 16 h, the morphology of embryonic neu- ron cells was observed by mi- croscopy. c Embryonic neuron cells were transiently transfected with green fluorescent protein (GFP) plasmid and stimulated with 1 μM of TLR9 ligands, ODN 2006, or ODN 2216, 72 h thereafter. The morphology and neurite length of embryonic neuron cells were observed by microscopy and traced by Image J software. d The neurite length traced was also quantified by Image J software. Data represent the mean±SD; **P<0.01. e Em- bryonic hippocampal neuron cells were transiently transfected with GFP-fused TLR9 siRNA plasmid or psi-GL3 empty vector as indicated. The knock-down of TLR9 was confirmed by western blotting. Actin was used as a loading control. f Cells treated as described in (e) were stimulated with 1 μM of ODN 2216 72 h thereafter, and the cell morphol- ogy and neurite length were ob- served and traced as described above. The quantification of neurite length is shown in (g). Data represent the mean±SD; ***P<0.001; *P<0.05 NS not significant

Article Snippet: Reagents TLR9 ligand, CpG-AODNs (ODN1585 andODN2216), CpGB ODNs (ODN1668 and ODN2006), and their controls were purchased from InvivoGen (San Diego, CA). psiGL3 vector (silence control vector) and psi-mTLR9 (mTLR9 silenced plasmid) were also purchased from InvivoGen (San Diego, CA).

Techniques: Isolation, Purification, Expressing, Western Blot, Control, In Vitro, Transfection, Plasmid Preparation, Microscopy, Software, Knockdown

Journal: Molecular neurobiology

Article Title: 3'poly-G-tailed ODNs inhibit F-spondin to induce cell death and neurite retraction in rat embryonic neurons.

doi: 10.1007/s12035-012-8275-8

Figure Lengend Snippet: Fig. 4 YB-1 deletion NLS mutant (YB-1-dNLS) attenuates 3′G-ODNs-mediated cytotoxicity and caspase activity in embryonic neuron cells. a Subcellular localization of YB-1 and YB-1-dNLS in neuro-2a cells. Neuro-2a cells were trans- fected with either YB-1 or YB-1- dNLS plasmid construct. Locali- zation of YB-1 in neuro-2a cells was detected by GFP fluores- cence. The nuclei of cells were labeled with DAPI (blue). b Embryonic neuron cells were transfected with YB-1 or YB-1- dNLS plasmid for 48 h, and cul- tured for another 48 h in neuro- basal medium in the presence of 2 μM 3′G-ODN. MTS assay was used to determine cell viability. c Percentage of damaged embry- onic neuron cells was detected by using propidium iodide (PI) staining after 48-h incubation with 2 μM 5′G-ODN or 3′G- ODN. d The caspase 3/7 activi- ties in the lysates of empty vector (EV), YB-1, and YB-1-dNLS- transfected embryonic neuron cells were estimated after stimu- lation with or without 2 μM 3′G- ODN for 48 h as indicated. Data represent the mean±SD; N03; **P<0.01; NS not significant

Article Snippet: Reagents TLR9 ligand, CpG-AODNs (ODN1585 andODN2216), CpGB ODNs (ODN1668 and ODN2006), and their controls were purchased from InvivoGen (San Diego, CA). psiGL3 vector (silence control vector) and psi-mTLR9 (mTLR9 silenced plasmid) were also purchased from InvivoGen (San Diego, CA).

Techniques: Mutagenesis, Activity Assay, Plasmid Preparation, Construct, Labeling, Transfection, MTS Assay, Staining, Incubation

Journal: Clinical and Molecular Allergy : CMA

Article Title: Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells

doi: 10.1186/s12948-021-00163-8

Figure Lengend Snippet: Regulation of allergen uptake of pDC varies among different allergens. PDC isolated from fresh peripheral blood or buffy coats by BDCA-4 positive magnetic bead selection were cultured overnight in AIM-V medium containing 10 ng/ml IL-3 in the presence or absence of CpG ODN 2006 (2,5 µg/ml), IL-4 (500 U/ml) or IL-13 (100 U/ml) or allergen as indicated. Prior to incubation, allergens were labeled using an Alexa Fluor 555 staining kit as described in Material and Methods. Allergen concentrations were 20 µg/ml for Der p 1 and 10 µg/ml for OVA and Api. After incubation, surface staining was performed with FITC conjugated Lin-1 and PerCP Cy5.5 conjugated CD123 and allergen uptake was analyzed by flow cytometry. PDC were defined as Lin-1 − /CD123 ++ cells with pDC comprising 24–50%. A Dot plots show examples of allergen uptake regulation, which are summarized in ( B – D ), where box plots show percentages of allergen-positive pDC, central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. B n = 6, C n = 5, D n = 8 experiments. Significance levels were assessed by Friedman test with p-values: ** p < 0.005 and * p < 0.05

Article Snippet: The TLR9 agonist CpG oligodeoxynucleotide ODN 2006 (CpG B) with the specific sequence 5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’ and the cytokines IL-3, IL-4 and IL-13 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Isolation, Selection, Cell Culture, Incubation, Labeling, Staining, Flow Cytometry

Journal: Clinical and Molecular Allergy : CMA

Article Title: Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells

doi: 10.1186/s12948-021-00163-8

Figure Lengend Snippet: ODN 2006 suppresses uptake of Der p 1, but not Api in healthy and allergic subjects. PBMC from fresh blood samples were enriched for panDC and cultured overnight in AIMV medium containing 20 ng/ml IL-3 in the presence or absence of CpG ODN 2006 (2,5 µg/ml), IL-4 (500 U/ml) or IL-13 (100 U/ml) or allergen as indicated. Prior to incubation allergens were labeled using an Alexa Fluor 555 staining kit as described. Allergen concentrations were 20 µg/ml for Der p 1 and 10 µg/ml for bee venom. After incubation, allergen uptake was analyzed using flow cytometry. Frequencies of pDCs in the DC-enriched samples reached 24–50% as defined by Lin-1 − /CD123 ++ staining. A Results from healthy controls without house dust mite (HDM) allergy (white boxes, n = 6) and individuals allergic to HDM (grey boxes, n = 5) are shown. B Data from healthy controls without bee allergy (white boxes, n = 8) and individuals allergic to bee venom (grey boxes, n = 12) are shown. Box plots illustrate percentages of allergen-positive pDC, central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance levels were **p < 0.005 and *p < 0.05, assessed by Friedman test, followed by Dunn’s multiple comparisons test

Article Snippet: The TLR9 agonist CpG oligodeoxynucleotide ODN 2006 (CpG B) with the specific sequence 5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’ and the cytokines IL-3, IL-4 and IL-13 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Cell Culture, Incubation, Labeling, Staining, Flow Cytometry