Journal: PLoS ONE

Article Title: Sphingosine Kinase-2 Maintains Viral Latency and Survival for KSHV-Infected Endothelial Cells

doi: 10.1371/journal.pone.0102314

Figure Lengend Snippet: ( A, B ) pDMVEC were incubated with or without purified KSHV for 2 h. After an additional 24 h, cells were incubated with the indicated concentrations of ABC294640 (ABC) or vehicle for another 24 h, then ceramide and dihydro-ceramide (dh-ceramide) species quantified as described in Methods. ( C, D ) Cells were treated as (A), then the concentrations of intracellular (C) or extracellular (D) S1P was quantified by ELISA. Error bars represent the S.E.M. for three independent experiments. * = p<0.01.

Article Snippet: Concentrations of S1P in culture supernatants and pDMVEC cell lysates were determined using the S1P Assay Kit (Echelon) according to the manufacturers’ instructions.

Techniques: Incubation, Purification, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

doi: 10.1371/journal.pone.0011512

Figure Lengend Snippet: Oral keratinocytes were incubated with or without Sphk-1 inhibitor (2 µM) for 2 h prior to challenging the cells with various TLR agonists namely, heat inactivated P. gingivalis (MOI:100), FSL-1 (1 µg/ml), Pam3CSK4 (0.5 µg/ml), P.gingivalis LPS (1 µg/ml), E. Coli LPS (1 µg/ml), ssRNA (0.1 µg/ml), Poly I:C (5 µg/ml) ODN (0.5 µg/ml), Imiquimod (0.1 µg/ml), Flagellin (0.25 µg/mL) ( A ); the IL-1α (2.5 ng/ml), IL-1β (2.5 ng/ml) and TNF-α (2.5 ng/ml) ( B ) and GPCR agonist S1P (100 nM) ( C ) for 24 h. The supernatant was collected after 24 h and HBD-2 ELISA was performed using Human BD-2 ELISA kit. The Sphk-1 inhibitor ablated HBD-2 induction with the agonists tested. The time course experiment was performed by pretreating the cells with Sphk-1 (2 µg/ml) for 2 h before challenging with FSL-1 (1 µg/ml) for 0, 30, 60, 90, 120 and 240 min. The total protein was collected and subjected to immunoblot with ser225 phospho specific Sphk-1 antibody and β-actin as loading control. We noted increase in the phosphorylation level of Sphk-1 at ser225 as early as 60 min and the level of phosphorylation was down regulated in the presence of Sphk-1 inhibitor ( D ). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).

Article Snippet: TransAM NF-kB p65 kit was purchased from Active Motif, CA and the ELISA kit for S1P was procured from Echelon Biosciences, UT, HBD-2 ELISA kit was from Peprotech, NJ and IL-6, IL-1β and TNF-α were from BD biosciences, CA.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

doi: 10.1371/journal.pone.0011512

Figure Lengend Snippet: The cells were transiently transfected with siRNA against Sphk-1 and incubated with FSL-1 (1 µg/ml), Imiquimod (0.1 µg/ml) and IL-1β (2.5 ng/ml). The supernatant was collected after 24 h challenge and subjected to human HBD-2 ELISA. siRNA against Sphk-1 down modulated agonist induced HBD-2 in HGECs ( A ). Intracellular S1P was measured using S1P ELISA kit after challenging with FSL-1 (1 µg/ml) for 2 h in cells pre-incubated with Sphk-1 inhibitor. The reaction was terminated after 2 h and 100 µg of total protein was subjected to ELISA. The intracellular S1P production was downregulated by Sphk-1 inhibitor ( B ). Total RNA was collected and converted to cDNA from the cells challenged with FSL-1 (1 µg/ml) for 24 h. cDNA was subjected to real time PCR with Sphk-1, Sphk-2 and GAPDH endogenous control TaqMan probes. Sphk-1 mRNA expression was highly upregulated compared to Sphk2 mRNA expression showing Sphk-1 as a predominant kinase in HGECs upon ligand challenge ( C ). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).

Article Snippet: TransAM NF-kB p65 kit was purchased from Active Motif, CA and the ELISA kit for S1P was procured from Echelon Biosciences, UT, HBD-2 ELISA kit was from Peprotech, NJ and IL-6, IL-1β and TNF-α were from BD biosciences, CA.

Techniques: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing