Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: Inhibition of SK2 expression inhibits CHIKV infection. ( A and C ) U2OS cells were transfected with a siRNA control (siCTRL) (50 nM) ( A ), SK2 (50 nM) siRNA ( A ), siCTRL (15 nM) ( C ) or SK1 siRNA (15 nM) ( C ). At 48-h post-transfection, knockdown was confirmed for SK2 ( B ) and SK1 ( D ) by qRT-PCR. In parallel the cells were infected with CHIKV at a MOI of 5 and at 48-h postinfection the cells were fixed and analyzed by high-content confocal microscopy. ( E ) HeLa cells were untreated or transfected with siCTRL or siSK2 then infected with CHIKV, as previously mentioned. At 48-h postinfection, supernatant was collected and viral RNA copy number determined by qRT-PCR analysis. Values are means + SEM ( n = 4). * P <0.0005.

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Inhibition, Expressing, Infection, Transfection, Control, Knockdown, Quantitative RT-PCR, Confocal Microscopy

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: SK2 inhibition impairs CHIKV infection. ( A and B ) HepG2 cells were pretreated for 2 h with 5, 10, 20, or 30 µM of SKI-II ( A ) or ABC ( B ) then infected with CHIKV at an MOI of 5. At 48-h postinfection, cells were fixed and analyzed for infection by high-content confocal microscopy. Values represent mean + SEM ( n = 3). * P < 0.05, ** P < 0.01 ( C ) HeLa cells were pretreated with either 50, 100, or 500 nM S1P for 15 min then infected with CHIKV at a MOI if 5 for 24 h. Cell lysates were harvested and subjected to Western blot analysis to detect the E2 viral glycoprotein and GAPDH, the relative intensity for each band is shown below.

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Inhibition, Infection, Confocal Microscopy, Western Blot

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: SK2 is re-localized during CHIKV infection. HeLa ( A ), hSKMC ( B ), or MCF10A ( C ) cells were uninfected or infected with CHIKV at a MOI of 5 for 24 h. The viral E2 protein (green), SK2 (red), and Hoechst nuclear stain (blue) were visualized by confocal microscopy. Scale bar = 50 µm ( A ), 10 µm ( B ), 25 µm ( C ).

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Infection, Staining, Confocal Microscopy

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: SK2 is required for an early step in virus infection. HeLa cells were treated with 20 µM of ABC at 2 h prior to infection or, 2-, 4-, or 6-h postinfection. The cells were then infected at a MOI of 5 for 48 h then infection analyzed by high-content confocal microscopy.

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Virus, Infection, Confocal Microscopy

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: Identification of SK2 interaction partners during CHIKV infection. Network interaction map showing SK2 interactions gene expression network clusters. Network plots were generated using the Cytoscape 3 network analysis tool. Color codes represent, SK2 (white), proteins that directly interact with SK2 by AP-MS (green), secondary interactions of proteins associated with mRNA processing and gene expression (blue).

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Infection, Gene Expression, Generated, Protein-Protein interactions

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: SK2 associates with the CHIKV RNA and nsP2 during infection. ( A ) BHK-21 cells were infected with CHIKV at a MOI of 5 for 1 or 2 h or mock infected, then fixed and stained for dsRNA (green), SK2 (red), and Hoechst nuclear stain (blue) and visualized by confocal microscopy. Scale bars = 2.5 µm. ( B ) HeLa cells were mock or infected with CHIKV at a MOI of 5. At 4-h postinfection, the cells were treated with ActD then subsequently treated with 5-EU. Labeled RNA was subsequently detected with an Alexa Fluor 488-coupled azide. Labeled RNA (green), viral E2 protein (cyan), SK2 (red), Hoechst nuclear stain (blue) were visualized by confocal microscopy. Scale bars = 5 µm. ( C ) BHK-21 cells were either mock infected or infected with CHIKV at an MOI of 10 for 2 h. The cells were fixed and immune-stained for nsP2 (green), SK2 (red), and Hoechst nuclear stain (blue). Scale bars = 7.5 µm.

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Infection, Staining, Confocal Microscopy, Labeling

Journal: Emerging Microbes & Infections

Article Title: Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

doi: 10.1038/emi.2015.61

Figure Lengend Snippet: CHIKV nsP3 co-localizes with SK2. ( A and B ) HeLa cells were transfected with the indicated expression plasmids for GFP-fused CHIKV nsP1, nsP2, nsP3, nsP4, and capsid. At 24-h post-transfection, the cells were fixed and immunostained for GFP-fused viral proteins (green) SK2 (red) and Hoechst nuclear stain (blue). ( B ) GFP-nsP3 transfected cells were co-stained for SK2 (red), G3BP (cyan), and Hoechst nuclear stain (blue). Scale bars = 7.5 µm ( A ), 5 µm ( B ).

Article Snippet: The pCMV6/Myc-DDK SK2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Transfection, Expressing, Staining

Journal: Endocrinology

Article Title: Sphingosine kinase as a regulator of calcium entry through autocrine sphingosine 1-phosphate signaling in thyroid FRTL-5 cells.

doi: 10.1210/en.2009-0288

Figure Lengend Snippet: FIG. 6. Calyculin A-evoked calcium entry in cells transduced with wild- type SK1, the G82D dominant-negative SK1 mutant, or the wild-type SK2. A, Cells transduced with wild-type SK1 (trace a) or the dominant- negative mutant G82D of SK1 (trace c) were pretreated with 100 nM calyculin A, and 1 mM calcium was added. Trace b shows calyculin A- evoked calcium entry in mock-transduced cells. Each trace is representative of at least seven separate determinations. B, Summary of several experiments performed as described in A. Each bar gives the mean SEM of seven to 14 determinations. *, P 0.05. C, G82D cells were treated with 100 nM calyculin A, and 1 mM calcium was added (trace a). In trace b, 100 nM S1P was added to calyculin A-treated cells before the start of the measurement. Trace c indicates calyculin- evoked calcium entry in cells transduced with wild-type SK1. D, Summary of several experiments performed as described in C. The bar denoted G82DS1P indicates the restoration of calcium entry in calyculin A-treated G82D cells after addition of S1P, and wt indicates the calcium entry in wild-type transduced cells treated with calyculin before addition of calcium. Each bar gives the mean SEM of five to eight determinations. *, P 0.05 compared with wt; §, P 0.05 compared with G82D. E, Cells transfected with plasmid only (trace a) or wild-type SK2 (trace b) were treated with calyculin A, and 1 mM calcium was added. The traces are representative of six separate experiments. F, Simplified scheme for how Sph, S1P, and SK1 may regulate calcium entry in FRTL-5 cells, and the role of calyculin A (Caly A). PPA denotes a presently uncharacterized phosphatase. We cannot exclude the possibility that calyculin also blocks a phosphatase directly regulating the calcium entry pathway.

Article Snippet: Transient transfection of FRTL-5 cells with SK2 The expression vector pCMV6-XL4-SphK2 was bought from Origene (Rockville, MD).

Techniques: Transduction, Dominant Negative Mutation, Mutagenesis, Transfection, Plasmid Preparation