Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Comparison of the effects of Cdt on toxicity in Jurkat and HUT78 cells. Jurkat cells were treated with medium alone (panel A) or Cdt (2.0 pg/ml) for 16–20 hr and cell cycle distribution determined following staining with propidium iodide (PI) and analysis by flow cytometry. Panels C and D show HUT78 cells in the presence of medium alone or Cdt, respectively. In addition to cell cycle, the effects of Cdt on apoptosis was assessed using the TUNEL assay; Panels E and F show the results from Jurkat cells in the presence of medium and Cdt (25 pg/ml), respectively, 48 hr after exposure to toxin. Results with HUT78 cells are shown in panels G (medium only) and H (Cdt). Numbers in each panel represent the percentage of G2 cells or apoptotic cells. Panels I and J show the aggregate results from three experiments each performed in duplicate; *indicate P<0.05 when compared to control Jurkat cells and ** indicates P<0.05 when compared to Cdt-treated Jurkat cells.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Comparison, Staining, Flow Cytometry, TUNEL Assay, Control

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Baseline analysis of components of the PI-3K signaling pathway. Panel A shows a representative Western blot for pAkt, Akt, pGSK3β (pGSK), GSK3β (GSK) and GAPDH for Jurkat cells, CCRM-CEM cells and HUT78 cells. Panel B shows the aggregate results for three experiments; results are expressed as a percentage of Jurkat cell density. Panel C shows the results of PIP3 levels and represent the mean SEM. *indicate P<0.05 when compared to control Jurkat cells

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Western Blot, Control

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Analysis of Jurkat cells reconstituted with PTEN. Panel A shows the baseline PIP3 levels were measured in Jurkat cells reconstituted with PTEN: controls (Con18) and pten transfection (PIJ-12); these cells were assessed under non-induced and induced conditions. Values are pmol PIP3/106 cells and represent the mean ± SEM of three replicate experiments; *indicate P<0.05 when compared to uninduced cells treated with Cdt. Panel B shows the effect of Cdt holotoxin on PIJ-12 and Con 18 cell lines which were treated with doxycycline as well as cells maintained under non-induced conditions. The percentage of G2 cells is plotted versus Cdt concentration. The mean ± SEM is shown for three experiments; *indicate P<0.05 when compared touninduced cells treated with Cdt.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Transfection, Concentration Assay

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Flow cytometric analysis of the binding of Cdt holotoxin and internalization of CdtB in Jurkat cells and HUT78 cells. Jurkat cells (panels A and B) and HUT78 cells (panels C and D) were exposed to medium (panels A and C) or Cdt (panels B and D). Cells were then stained for surface association of Cdt holotoxin (anti-CdtC mAb) or internalization of CdtB (anti-CdtB mAb). The mean channel fluorescence (MCF) is indicated in each panel for both control cells not exposed to Cdt (grey curve) and toxin treated cells (black line). The mean MCF ± SEM for three experiments is shown in panel E; *indicate P<0.05 when compared to control cells.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Binding Assay, Staining, Fluorescence, Control