b0304  (Echelon Biosciences)

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A. PI3K inhibitors reduce plaque size in BSC40 cells. Cells were infected with 100 PFU of vaccinia virus (VV), strain WR. Inhibitors were added one hour post viral entry. First row: LY294002, 20 µM; B0304, 20 µM; AS1, 50 µM; AS2, 50 µM (LY294002 and B0304 are in 0.2% DMSO and AS1 and AS2 are in 0.5% DMSO); 0.5% DMSO alone. Insets show representative plaques from 50 µM AS1 and 0.5% DMSO treated wells. Second row: 10 µM Wortmannin in 0.1% DMSO did not appear to reduce VV plaque size; 0.1% DMSO control. Results are from three independent trials. B. p85WT and p85α −/− cells form two times more plaques than p85α −/− β −/− cells. Cells were infected with equal amounts of virus and monolayers were stained with crystal violet 48 hours post infection. Results are from eight independent trials. C. PI3K inhibitors do not decrease early viral protein expression. BSC40 cells were infected at pH 7.4 and MOI = 5 with VV and PI3K inhibitors were added 1 hour post-viral entry and allowed to infect for an additional 2 hours. Proteins were subjected to western analysis by anti-E3 mAb and anti-Tubulin mAb. For quantification (right) bands were normalized to tubulin loading control and data is expressed as the fold change relative to DMSO-treated samples. D. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV at pH = 7.4 and analyzed as in C. E. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV. Virus was bound to cells at 4°C, after which cells were moved to 37°C with PBS, pH = 5. Following a 2 hour infection, equal amounts of protein were subjected to western analysis with anti-E3 mAb or anti-Tubulin, and quantitated as in C.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Infection, Staining, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: 50% Inhibitory Concentration (IC 50 ) of PI3K inhibitors added post-adsorption to poxvirus-infected monolayers.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A. PI3K inhibitors reduce plaque size in BSC40 cells. Cells were infected with 100 PFU of vaccinia virus (VV), strain WR. Inhibitors were added one hour post viral entry. First row: LY294002, 20 µM; B0304, 20 µM; AS1, 50 µM; AS2, 50 µM (LY294002 and B0304 are in 0.2% DMSO and AS1 and AS2 are in 0.5% DMSO); 0.5% DMSO alone. Insets show representative plaques from 50 µM AS1 and 0.5% DMSO treated wells. Second row: 10 µM Wortmannin in 0.1% DMSO did not appear to reduce VV plaque size; 0.1% DMSO control. Results are from three independent trials. B. p85WT and p85α −/− cells form two times more plaques than p85α −/− β −/− cells. Cells were infected with equal amounts of virus and monolayers were stained with crystal violet 48 hours post infection. Results are from eight independent trials. C. PI3K inhibitors do not decrease early viral protein expression. BSC40 cells were infected at pH 7.4 and MOI = 5 with VV and PI3K inhibitors were added 1 hour post-viral entry and allowed to infect for an additional 2 hours. Proteins were subjected to western analysis by anti-E3 mAb and anti-Tubulin mAb. For quantification (right) bands were normalized to tubulin loading control and data is expressed as the fold change relative to DMSO-treated samples. D. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV at pH = 7.4 and analyzed as in C. E. Early protein expression is reduced in p85-deficient cells. Cells were infected at an MOI = 5 with VV. Virus was bound to cells at 4°C, after which cells were moved to 37°C with PBS, pH = 5. Following a 2 hour infection, equal amounts of protein were subjected to western analysis with anti-E3 mAb or anti-Tubulin, and quantitated as in C.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Infection, Staining, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: 50% Inhibitory Concentration (IC 50 ) of PI3K inhibitors added post-adsorption to poxvirus-infected monolayers.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: BHK cells were infected with VV at an MOI of 5, and PI3K inhibitors were added following entry. After 16 hours proteins were subjected to western analysis with various antibodies, including anti-Tubulin to ensure equal loading. Quantitation (right) is described in . Data are expressed as the fold change relative to DMSO-treated samples. A. Cells were infected with VV strain WR expressing B5-GFP under its own promoter, and subjected to western analysis with GFP (top) or tubulin (bottom) antibodies. B. Cells were infected with VV strain WR expressing F13-GFP, and analyzed as in A. C . Cells were infected with VV strain WR for 6 or 16 hours. Filters were probed with P4b/4b or tubulin antibodies. Results are from two or three independent trials.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Infection, Western Blot, Quantitation Assay, Expressing

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A . BHK cells were infected with VV F13-GFP, and treated with AS1 (50 µM) or AS2 (50 µM) post-adsoption for 16 hours. Cells were fixed and stained with phalloidin to visualize actin (red) and DAPI to visualize DNA (blue). Approximately 600 cells were counted per condition and cells were scored positive if a FITC (GFP) signal could be detected. B. BHK cells were infected with VV B5-GFP, and treated with PI3K inhibitors post adsoption for 16 hours, and stained as in A. Approximately 400 cells were counted per condition. C. PI3K inhibitors alter the distribution of F13-GFP in infected cells. Cells were infected and stained as in A. Note that AS1 and AS2 cause a reduction of punctate FITC fluorescence at the cell periphery, which represents virions, as well as a reduction in the number of actin tails. AS1 also caused F13-GFP to localize to the cytoplasm instead of on punctate virions. D. PI3K inhibitors alter the distribution of B5-GFP in infected cells. Cells were infected and stained as in B. Note that AS1 and AS2 had similar effects on localization of B5 and F13, namely a reduction in peripheral punctate virions and reduced numbers of actin tails. Results are from three independent trials.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Infection, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A-B. Multistep growth curves of cell-associated (A) or released virus (B) conducted at an MOI of 0.01. AS1 and AS2 were used at 50 µM, Rifampicin 0.1 mg/mL, and the carrier DMSO at 0.5%. A. Multistep growth curves of cell-associated infectious virions, MOI of 0.01. AS1 and rifampicin reduce the amount of virus produced over time relative to DMSO control cells, (20.8-fold and 15.4-fold reduction at 36 hours). AS2 caused a modest reduction in the amount of virus produced over time relative to DMSO controls (5-fold reduction at 36 hours). B. Multistep growth curves of released infectious virions, MOI of 0.01. Note that AS1, AS2, and rifampicin reduce the amount of virus released into the supernatant (Fold reduction at 36 hours: AS1- 127.8-fold, AS2- 28.8-fold, Rifampicin- 55.6-fold). C-D. Multistep growth curves of cell-associated or released virus conducted at an MOI of 0.01 in p85-deficient cells. C. Multistep growth curves of cell-associated infectious virions, MOI of 0.01. From 8 to 36 hours, a reduction in the amount of virus produced was apparent in p85α −/− β −/− cells compared to the p85WT and p85α −/− cells (19.3-fold reduction at 36 hours). D. Multistep growth curves of released infectious virions, MOI of 0.01. A reduction in the amount of virus released from the p85α −/− β −/− cells compared to the p85WT and p85α −/− cells was apparent (20-fold reduction at 36 hours). Results are from one trial conducted in triplicate, and match growth curves conducted using WR. E. PI3K inhibitors reduce comet tails formed by VV, strain IHD-J. Monolayers were infected with 100PFU of virus, and plaques were visualized 48 hours later with crystal violet stain. Experiments were conducted in three independent trials. F,G. Toxicity assays of PI3K inhibitors on uninfected cells. F. MTS assay on BSC-1 cells treated with PI3K inhibitors for different periods of time. MTS production is a measure of mitochondrial metabolism. Values represent the concentration of drug (in µM) required to reduce formazan production by half. Experiments were conducted in two independent trials. G. Trypan blue exclusion assays of BHK cells treated with PI3K inhibitors (AS1 and AS2, 50 µM) for different time periods.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Produced, Infection, Staining, MTS Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A. BHK cells were infected with MVA for 17 hours plus or minus PI3K inhibitors. Untreated samples exhibit characteristic virion morphogenesis (4100x, Glut/KMnO 4 fixative). A′: 1) Crescents; 2) Immature virions; 3) Mature virion, IMV; A″: 4) IEV; 5) CEV; 6) EEV. B. In cells treated with 50 µM AS1, virions at the immature to IMV transition, but not at later stages, are evident (10,000x, Glut/OsO 4 fixative). a) Replication center and crescents; b) Immature virion; c) Immature virion with nucleoid. C. In cells treated with 50 µM AS2, virions at the IMV wrapping step but not at later stages are evident (6800x, Glut/KMnO 4 Fix). a') Immature virion; b') IMV. Note the lack of virions associated with the plasma membrane, characteristic of IEV inhibition. D. HeLa cells were infected with B5-GFP WR for 17 hours plus or minus PI3K inhibitors. Untreated samples exhibit characteristic virion morphogenesis (10,000x, Glut/KMnO 4 Fix). D': 1) Crescents; 2) Immature virions; D″: 3) Mature virion, IMV; 4) IEV. E. In cells treated with 50 µM AS1, virions at the immature to IMV transition, but not at later stages, are evident (10,000x, Glut/OsO 4 Fix). a) Viral crescents; b) Immature virion; c) Immature virion with nucleoid. F. Samples treated with 50 µM AS2 are stuck at the IMV wrapping step (7000x, Glut/KMn 4 ). a′) Crescents; b′) Immature virion; c′) IMV. Note the lack of virions associated with the plasma membrane, a characteristic of IEV inhibition.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Infection, Inhibition

Journal: PLoS ONE

Article Title: Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

doi: 10.1371/journal.pone.0010884

Figure Lengend Snippet: A. PI3K inhibitors reduce plaque numbers in p85-deficient cells. Drugs were added to p85-deficient cells post viral adsorption at different concentrations. The concentration of drug needed to reduce plaque numbers by half (IC 50 ) was calculated by fitting the data to a linear regression model using Prism software (GraphPad Prism Software, Inc., La Jolla, CA). IC 50 values are lower for p85-deficient cells. Results are from three independent trials. B. Summary of vaccinia virus morphogenesis in p85-deficient cells or following PI3K inhibitor treatment. After VV entry (1) virion uncoating and early protein synthesis occurs (2). These steps are followed by late protein production (3), formation of viral replication centers and viral crescents (4). Crescents envelope viroplasm to form immature virions (IV, 5). The cores of IV condense to form mature virions (IMV, 6). A subset of the IMV traffic to the Golgi Complex where they are enveloped in a host cell derived membrane to form IEV (7). IEV fuse with the plasma membrane, releasing virus to the extracellular milieu, (CEV, 8). CEV form actin tails beneath the virus and ultimately, release to form extracellular enveloped virions (EEV, 9). Our results suggest that involvement of PI3Ks at steps 2,3,5, and 7. Steps 2 and 7 are disrupted in the p85-deficient cells, AS1 treatment disrupts steps 3 and 5, and AS2 disrupts steps 7. Thus, PI3K usage by VV is redundant; multiple PI3Ks acting at each of several steps regulate VV morphogenesis.

Article Snippet: The PI3K-alpha Inhibitor 2 #B0304 or 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, referred to as B0304, was obtained from Echelon Biosciences, and LY294002 was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Adsorption, Concentration Assay, Software, Derivative Assay

Journal: Cell reports

Article Title: Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration

doi: 10.1016/j.celrep.2019.12.083

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PI3K p110α inhibitor (PIK2) , Echelon Biosciences Inc. , Cat#B-0304.

Techniques: Recombinant, Selection, Cell Isolation, Software

Journal: Scientific Reports

Article Title: An essential role of PI3K in the control of West Nile virus infection

doi: 10.1038/s41598-017-03912-5

Figure Lengend Snippet: Selective inhibition of the PI3K catalytic p110 δ reduces antiviral immune responses. ( A ) The mRNA abundances of Ifnb1 , Tnfa and p110 isoforms during the course of WNV infection were quantified by q-PCR. The results are expressed as fold change over uninfected (0 h after infection). ( B–D ) BMDMs were left untreated (mock) or infected with WNV in the presence of 5 µM of a pan-PI3K inhibitor LY294002 (All) or various concentrations of p110 isoform-specific inhibitors for 24 h. None: no inhibitor. ( B ) The cellular viral loads were quantified by q-PCR. The data are expressed as fold change over None. ELISA of ( C ) IFN-β and ( D ) TNF-α levels in the cell culture media. *p < 0.05, **p < 0.001 (unpaired Student’s T-test), Bar: the mean of the result + s.e.m. The data shown are representative of 3 independent reproducible experiments.

Article Snippet: The PI3-Kinase p110 α subunit inhibitor (B-0304, IC50 = 2 nM)), and γ inhibitor B-0302(IC50 = 250 nM) were from the Echelon (Salt Lake City, UT 84108, USA).

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Scientific Reports

Article Title: An essential role of PI3K in the control of West Nile virus infection

doi: 10.1038/s41598-017-03912-5

Figure Lengend Snippet: siRNA knock down of the PI3K catalytic p110 δ reduces antiviral immune responses. The scrambled (Neg) or p110-targeting siRNA was electroporated into BMDMs, 48 h later the BMDMs were infected with WNV for 24 h. ( A , B ) The mRNA abundances of p110 isoforms were quantified by q-PCR after WNV infection. The results are expressed as fold change over Neg in ( A ) or Mock transfected in ( B ). ( C ) The cellular viral loads were quantified by q-PCR. The data are expressed as fold change over Neg. ( D ) ELISA of IFN-β in the cell culture medium. UI, uninfected. *p < 0.05 (unpaired Student’s T-test), Bar: the mean of the result + s.e.m. The data shown are representative of 3 independent reproducible experiments.

Article Snippet: The PI3-Kinase p110 α subunit inhibitor (B-0304, IC50 = 2 nM)), and γ inhibitor B-0302(IC50 = 250 nM) were from the Echelon (Salt Lake City, UT 84108, USA).

Techniques: Infection, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture