Journal: Science signaling

Article Title: The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P

doi: 10.1126/scisignal.ade3643

Figure Lengend Snippet: PI4KB and OSBP inhibition have opposite effects on STING activation A. THP-1 cells were pre-incubated with the PI4KB inhibitor BF738735 and subsequently stimulated with the STING agonist 2’3’-RR CDA and tdTomato-reporter expression was quantified by flow cytometry. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. A paired one-tailed t-test was performed on data that was not normalized to the controls. B. PI4KB mRNA expression levels in THP-1 cells expressing a control gRNA or gRNAs targeting PI4KB. Mean ± SEM of n = 4 biological replicates out of 4 independent experiments are shown. One-sample t-tests were performed to compare each group to the normalized control value set at 1. C. Cells in (b) were stimulated with 2’3’-RR CDA and tdTomato reporter expression was quantified by flow cytometry. Mean ± SEM of n = 5 biological replicates out of 5 independent experiments are shown. Statistical tests were performed on unnormalized data. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare each treatment group to the control group. D. 293T cells expressing eGFP-mSTING were transfected with a phosphatase-dead Sac1 (inactive), active Sac1 wt, or Sac1-kkaa mutant and stimulated or not with 2’3’-RR CDA (RR-CDA). After stimulation, cells were stained for phospho-STING and analyzed by flow cytometry. Mean ± SEM of n = 4 biological replicates out of 4 independent experiments are shown. One-sample t-tests were performed to compare each group to the normalized control value set at 100. E. THP-1 cells were preincubated with DMSO or the OSBP inhibitors itraconazole (ITZ) or OSW-1 and stimulated with 2’3’-cGAMP or left untreated. Reporter expression was quantified by flow cytometry. Representative images of n = 3 biological replicates out of 3 independent experiments are shown. F. Mean fluorescence intensity of tdTomato reporter in THP-1 cells stimulated with 2’3’-cGAMP in the presence of DMSO, itraconazole (ITZ), or OSW-1. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. Statistical tests were performed on unnormalized data. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare each treatment group to the control group. G. CXCL10 mRNA levels in THP-1 cells pre-treated with DMSO or itraconazole (ITZ) and stimulated with 2’3’-RR CDA (RR-CDA). Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. A paired one-tailed t-test was used to compare the ITZ group to the DMSO control group. H. tdTomato reporter expression of THP-1 cells pre-treated with the PI4KB inhibitor (PI4KBi) BF738735 followed by pre-treatment with itraconazole (ITZ) and stimulation with 2’3’-cGAMP. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare the indicated treatment groups to the control group. * P< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: Reagents used include itraconazole (Santa Cruz Biotechnology cat. no. sc-205724A), OSW-1 (a kind gift from M. Shair, Harvard University), BF738735 (Tocris cat.no.

Techniques: Inhibition, Activation Assay, Incubation, Expressing, Flow Cytometry, One-tailed Test, Control, Transfection, Mutagenesis, Staining, Fluorescence

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–F) Vero cells were pretreated with DMSO (as the control) or the indicated concentrations of 25-HC (A), U18666A (B), or BF738735 (C) for 24 h and then transfected with AiV replicon RNA. At 10 h after transfection, the cells were analyzed for luciferase activity. Data were normalized to the DMSO treatment, and cell viability was scored. (D–F) Vero IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (–) or 1 μM of 25-HC (D), 1.5 μM of U18666A (E), or 150 nM of BF738735 (F). After 24 h, the cells were transfected with AiV replicon RNA, and luciferase activity was determined at 10 h after transfection. (G) Cell viability was measured in parallel. The maximum value obtained for Tet (–) drug-untreated cells was taken as 100%. Data are the mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: BF738735 was from Med Chem Express.

Techniques: Control, Transfection, Luciferase, Activity Assay, Incubation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–E) Vero-IFITM1 cells were incubated with or without Tet for 48 h and then treated with mock (A and D), 1 μM 25-HC (B), 1.5 μM U18666A (C), or 150 nM BF738735 (E), followed by pCMV-polyprotein transfection. At 24 h after transfection, the cells were fixed and stained with filipin III (A–C) or anti-PI4P (D and E), anti-IFITM1 and anti-2B antibodies. Bars, 4 μm. Pearson’s correlation coefficient was measured for individual cells.

Article Snippet: BF738735 was from Med Chem Express.

Techniques: Incubation, Transfection, Staining