Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells were infected with influenza A/WSN/33 (H1N1) virus (WSN) or ( B ) influenza A/PR/8/34 (H1N1) virus (PR8) at an MOI of 0.5 for the indicated times. The mRNA levels of SPHK2 were analyzed by qRT-PCR at 0, 6, 12, 24 and 48 hpi, which was normalized to GAPDH. Error bars represent mean values ± SD calculated from the results for three individual samples. **, P<0.01; ***, P<0.001; ****, P<0.0001. ( C ) A549 cells were infected with influenza A/WSN/33 (H1N1) virus (WSN) or influenza A/PR/8/34 (H1N1) virus (PR8) at an MOI of 0.5 for 24 h. The protein level of SPHK2 was measured by Western blotting. NP, M1 and NS1 were used as positive controls of IAV infection, and β-actin was an internal loading control.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Infection, Virus, Quantitative RT-PCR, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: A549 cells were uninfected or infected with influenza A/WSN/33 (H1N1) virus (WSN) at an MOI of 0.5 for 6 h. ( A ) Cells were fixed and stained using DAPI for nuclei (Blue), as well as anti-influenza NP (Green) and anti-SPHK2 antibodies (Red). The cells were visualized by confocal laser scanning microscopy. ( B ) The protein levels of SPHK2 and IAV NP in uninfected or infected A549 cells were detected by Western blotting, β-actin and Lamin B were used as loading controls for cytoplasmic and nuclear proteins, respectively. Results are representatives of three independent experiments.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Infection, Virus, Staining, Confocal Laser Scanning Microscopy, Western Blot

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells were transfected with plasmids encoding SPHK2 or a control vector pCMV-Myc, and 24 h after transfection, cells were infected with WSN virus at an MOI of 0.5. A549 cells or sgSPHK2-1 A549 cells were infected with WSN virus at an MOI of 0.5. At 24 hpi, Western blotting analysis was performed to detect viral NP, M1, NS1 and β-actin is shown as a loading control. ( B ) Virus titers in IAV (MOI = 0.5) infected A549 cells transfected with the plasmids pCMV-myc or Myc-SPHK2 and virus titers in IAV infected A549 cells or IAV infected sgSPHK2-1 A549 cells was determined in MDCK cells by plaque assay. Values are means ± SD (n = 3). Data are representative of three independent experiments. **, P<0.01; ****, P<0.0001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Transfection, Control, Plasmid Preparation, Infection, Virus, Western Blot, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) HDAC1 was stably knocked down in A549 cells. The mRNA and protein levels of HDAC1 was measured by qPCR and Western blotting respectively. ( B ) A549 cells were transfected with negative control (CTRL-siRNA) or SPHK2-targeting siRNAs (SPHK2-siRNAs) to silence SPHK2, and 48 h later, the mRNA and protein levels of SPHK2 was measured by qPCR and Western blotting respectively. ( C ) shHDAC1 A549 cells were transfected with siRNAs (CTRL-siRNA or SPHK2-siRNA-1, SPHK2-siRNA-2) and infected with WSN virus at an MOI of 0.5. After 24 h infection, the expression of NP, M1 and NS1 were detected by Western blotting, β-actin was as an internal loading control. ( D ) shCTRL A549 cells or shHDAC1 A549 cells were transfected with control vector or SPHK2-expressing plasmids and infected with WSN virus at an MOI of 0.5. After 24 h infection, the expression of NP, M1 and NS1 were detected by Western blotting. ( E ) shHDAC1 A549 cells were transfected with siRNAs (CTRL-siRNA or SPHK2-siRNA-1) and HDAC1 expression was restored. After 24 h, the cells were infected with WSN virus at an MOI of 0.5 and the expression of NP, M1 and NS1 were detected by Western blotting. ( F ) HDAC1 knock-down A549 cells (shHDAC1) were treated with CTRL-siRNA, SPHK2-siRNA-1 or transfected with Myc-SPHK2 plasmids, followed by restoration of HDAC1 expression. After 24 h, the cells were infected with WSN virus at an MOI of 0.5 and the virus titers were measured by plaque assay, and Myc-tagged HDAC1, SPHK2 protein in these cells were detected by Western blotting. Data are representative of three independent experiments. ns, no significance; **, P<0.01; ****, P<0.0001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Stable Transfection, Western Blot, Transfection, Negative Control, Infection, Virus, Expressing, Control, Plasmid Preparation, Knockdown, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) The amino acid sequence of SPHK2 wild-type (WT) and the mutation site of SPHK2-G212E are indicated. ( B and C ) A549 cells were transfected with vectors encoding Myc-tagged SPHK2-WT or SPHK2-G212E mutants, pCMV-Myc plasmid was as a control vector (-), and infected with WSN virus at an MOI of 0.5. After 24 h, Western blotting was used to detected the NP, M1 and NS1 protein (B). After 24 h infection, cell supernatants were collected for a plaque assay to measure the virus titers in A549 cells at each condition (C). All data are representative of three independent experiments showing similar results. ns, no significance; ***, P<0.001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Control, Infection, Virus, Western Blot, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A and B ) HEK293 cells were transfected with pCMV-Myc (-) or SPHK2-encoding plasmids, with luciferase plasmids (pGL3-IFNβ-promoter or pGL3-NF-κB-promoter with pRL-TK co-transfected into cells), and then infected with WSN virus at an MOI of 0.5. NF-κB (A) or IFN-β (B) activity was measured using luciferase reporter assays, and viral NP, M1, NS1 proteins in these cells were detected by Western blotting, β-actin was as an internal loading control. ( C ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5. IFN-β activity was measured using luciferase reporter assays, and viral NP, M1, NS1 proteins in these cells were detected by Western blotting. ( D ) A549 cells were transfected with pCMV-Myc (-) or SPHK2-expressing plasmids, and 24 h after transfection, cell were infected with WSN virus at an MOI of 0.5. The mRNA levels of IFIT1, ISG15 and IFNb was analyzed by qPCR at 24 hpi, which was normalized to the mRNA level of GAPDH. ( E ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5 for 24 h. The mRNA levels of IFIT1, ISG15 and IFNb was analyzed by qPCR at 24 hpi. ( F ) A549 cells were transfected with pCMV-Myc or Myc-SPHK2 plasmids, and 24 h after transfection, cells were infected with WSN virus at an MOI of 0.5. pSTAT1, STAT1, NP, M1 and NS1 protein in A549 cells were analyzed by Western blotting at 24 hpi. ( G ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5 for 24 h. pSTAT1, STAT1, NP, M1 and NS1 protein in A549 cells were analyzed by Western blotting at 24 hpi. Data are representative of three independent experiments. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Transfection, Luciferase, Infection, Virus, Activity Assay, Western Blot, Control, Knock-Out, Expressing

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells transfected with pCMV-Myc or Myc-SPHK2 were immunoprecipitated using anti-Myc antibody, and then Myc-tagged SPHK2, HDAC1, and TET3 proteins were analyzed by immunoblotting. ( B ) A549 cells transfected with pRK11-Flag or Flag-TET3 were immunoprecipitated using anti-Flag antibody, and then SPHK2, HDAC1, and Flag-tagged TET3 proteins were analyzed by immunoblotting. ( C ) A549 cells transfected with pCMV-Myc or Myc-HDAC1 were immunoprecipitated using anti-Myc antibody, and then SPHK2, Myc-tagged HDAC1, and TET3 proteins were analyzed by immunoblotting. ( D ) A549 cells uninfected or infected with WSN were immunoprecipitated using anti-Human SPHK2 polyclonal antibody (Proteintech, 17096-1-AP), and then SPHK2, HDAC1 and TET3 proteins were analyzed by immunoblotting. ( E ) Diagram of SPHK2-FL (full length) and deletion of SPHK2 fragments (F1, F2 and F3). ( F ) A 549 cells transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, and Myc-SPHK2-F3 were immunoprecipitated by using anti-Myc antibody, and then Myc-tagged SPHK2, Myc-tagged SPHK2-F3, HDAC1, and TET3 proteins were analyzed by immunoblotting. ( G ) HEK293 cells were transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, or Myc-SPHK2-F3, and then infected with WSN virus at an MOI of 0.5. IFN-β activity was measured by using luciferase reporter assays. ( H ) A549 cells transfected with pCMV-Myc (Mock), Myc-SPHK2 or Myc-SPHK2-F3, and IFN-β promoter enrichment for Myc-tagged SPHK2 or Myc-tagged SPHK2-F3 was detected by ChIP-qPCR. ( I ) IFN-β promoter enrichment for Myc-tagged HDAC1 in sgCTRL A549 cells or sgSPHK2-1 A549 cells overexpressed with Myc-tagged HDAC1 was detected by ChIP-qPCR. ( J ) A549 cells were transfected with CTRL-siRNA or TET3-siRNA, and 24 h post-transfection, cells were overexpressed with SPHK2. IFN-β promoter enrichment for Myc-tagged SPHK2 was detected by ChIP-qPCR. ( K ) A549 cells were uninfected or infected with WSN, and IFN-β promoter enrichment for SPHK2 was determined by ChIP-qPCR. Data are representative of three independent experiments. ns, no significance; **, P<0.01; ***, P<0.001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Activity Assay, Luciferase, ChIP-qPCR

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells and sgSPHK2-1 A549 cells were infected with IAV (WSN), MOI = 0.5, 24 h post infection, IFN-β promoter enrichment for H3ac, H4ac and HDAC1 antibodies was detected by ChIP-qPCR. ( B ) A549 cells transfected with pCMV-Myc or Myc-SPHK2 were infected with IAV (WSN), MOI = 0.5, 24 h post infection, IFN-β promoter enrichment for H3ac, H4ac and HDAC1 antibodies was detected by ChIP-qPCR. ( C ) A549 cells were treated with solvent (-) or TSA (5, 10, 20 or 50 nM) for 72 h, cell viability was detected by Cell Counting Kit-8. ( D ) A549 cells were treated with TSA (5, 10 and 20 nM), then transfected with Myc-SPHK2 and infected with WSN, MOI = 0.5, after 24 h, IFN-β mRNA level was measured by qPCR, and normalized to GAPDH. (E ) A549 cells were transfected with TET3-siRNA, then transfected with Myc-SPHK2 and infected with WSN, MOI = 0.5, after 24 h, cells IFN-β mRNA level was measured by qPCR. Data are representative of three independent experiments. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Infection, ChIP-qPCR, Transfection, Solvent, Cell Counting

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: The expression of SPHK2 in IAV infected cells is upregulated and then the location of SPHK2 is shifted from cytoplasm to nucleus. Afterwards, SPHK2 accumulated in the nucleus interacts with HDAC1 and its substrate binding domain also interacts with TET3 that bands to IFN-β promoter. Therefore, HDAC1 is recruited to IFN-β promoter and inhibited IFN-β transcription by enhancing the deacetylation of IFN-β promoter.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Expressing, Infection, Binding Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: Plasmid constructs used in the study.

Article Snippet: Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP).

Techniques: Plasmid Preparation, Construct, FLAG-tag, Sequencing, Mutagenesis