Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection

doi: 10.1073/pnas.1610197114

Figure Lengend Snippet: Schematics of the anti-HCV drug targets and the experimental system. (A) HCV life cycle and drug targets. After entry into the host cell, HCV genomic RNA is translated into viral precursor polyprotein and processed into functional proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV RNA replicates inside the isolated membrane compartments derived from the endoplasmic reticulum (ER), and assembles into viral particles on lipid droplets, which traffic through the Golgi and are released outside of the cell. PIs (TPV, DPV, ASV, and SMV) inhibit the processing step, and drugs such as NI (SOF), NNIs (VX, DAS, NSV, and TGV), NS5AIs (DCV and LDV), and CIs (CsA and SCY) target HCV RNA replication. IFNs (IFN-α and IFN-λ1) supposedly inhibit at least the step(s) of translation and replication. (B) HCV replication activity was evaluated using an HCV subgenomic replicon (genotype 1b, strain NN) carrying a fusion of the firefly luciferase gene (Luc) with the neomycin phosphotransferase (Neor). The replicon autonomously and persistently replicates in Huh-7 cells. Cells treated with drugs were incubated for 72 h and then harvested for luciferase assay. Inhibition of HCV replication was measured by the luciferase activity in drug-treated cells, relative to activity in DMSO-treated cells.

Article Snippet: SMV, ASV, DAS, NSV, TGV, and LDV were purchased from MedChem Express.

Techniques: Functional Assay, Isolation, Membrane, Derivative Assay, Activity Assay, Luciferase, Incubation, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection

doi: 10.1073/pnas.1610197114

Figure Lengend Snippet: Quantification of the IIP of single-HCV drugs. (A) Log–log plots of dose–response curves normalized by IC50, determined from the replicon assay, of PIs (TPV, DPV, SMV, and ASV; red), the NI (SOF; blue), NNIs (VX, DAS, NSV, and TGV; orange), NS5AIs (DCV and LDV; green), IFNs (IFN-α and IFN-λ1; cyan), and CIs (CsA and CSY; purple). Each point represents the mean of three experiments. (B) IIP of classes or subclasses of antiviral drugs, normalized by IC50, calculated from the experimentally measured fu by Eq. 1. (C) IIP values at drug concentration D = 100 × IC50 (IIP100) determined by extrapolation.

Article Snippet: SMV, ASV, DAS, NSV, TGV, and LDV were purchased from MedChem Express.

Techniques: Concentration Assay

Journal:

Article Title: Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30

doi: 10.1210/me.2008-0262

Figure Lengend Snippet: GPR30-mediated FN matrix assembly occurs independently of EGFR transactivation in SKBR3 breast cancer cells. A, SKBR3 cells were fed rat FN (rFN) (25 μg/ml) in the presence of either 17β-E2 (1 nm) or ICI 182,780 (1 μm) or no agonist (untreated, UN) for 18 h. Alternatively, cells were pretreated with the pan MMP-inhibitor, GM6001 (10 μm for 15 min), Src-like kinase inhibitor, PP1 (50 nm for 15 min), or CRM-197 (200 ng/ml for 1 h) before receiving rFN in the presence of 17β-E2. Equivalent amounts of DOC-soluble or -insoluble protein were blotted with rFN-specific mAB IC3. The immunoblot shown is a representative of four independent experiments. Band intensities from individual experiments were measured using Scion Image software. Mean integrated optical densities (±sem) were measured for unstimulated (28.6 ± 11.5) and 17β-E2-stimulated (148.1 ± 15.7) cells. B, SKBR3 cells were pretreated with either the erbB1-specific inhibitor, tyrphostin AG1478 (1 nm) or vehicle (DMSO) for 15 min before receiving rFN in the presence of 17β-E2 (1 nm), ICI 182, 780 (1 μm), EGF (10 ng/ml), HB-EGF (10 ng/ml), or no agonist. FN matrix assembly was measured after 18 h of treatment as described above. C, SKBR3 cells treated with AG1478 or vehicle as described in panel B in the absence of exogenous rFN. Cells were then lysed and erbB1 tyrosyl phosphorylation was assessed in erbB1 immunoprecipitates that were immunoblotted with phosphotyrosine-specific 4G10 mAB. ErbB1 recovery was determined by reprobing the same filter with sheep EGFR antibodies. DMSO, Dimethylsulfoxide.

Article Snippet: Src-family tyrosine kinase inhibitor, PP1, was purchased from AG Scientific Inc. (San Diego, CA).

Techniques: Western Blot, Software, Phospho-proteomics

Journal:

Article Title: Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30

doi: 10.1210/me.2008-0262

Figure Lengend Snippet: GPR30 signaling promotes the formation of Src-dependent, Shc-integrin α5β1 protein complexes. Triton X-100 extracts prepared from MDA-MB-231 cells stably expressing vector or GPR30 were left untreated (UN) or stimulated with 17β-E2, EGF, or pretreated with the Src family kinase inhibitor, PP1 (50 nm; 15 min) before stimulation with 17β-E2. Integrin α5β1 or α2β1 was immunoprecipitated with rabbit antibodies specific for the integrin α5 or α2 subunit cytoplasmic tails, respectively. Associated Shc proteins were detected with Shc mAB, PG797. Recovery of integrin α5β1 or α2β1 was determined by blotting with integrin α5 or α2 subunit protein antibodies. Relative positions of endogenous p66, p52, and p46 Shc are designated.

Article Snippet: Src-family tyrosine kinase inhibitor, PP1, was purchased from AG Scientific Inc. (San Diego, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: Serum-starved A549 cells were incubated (or not) with 1 µM AG1478 or 5 µM PP1 for 30 min. Then, the cells were treated for 30 min. with 100 ng/ml EGF or 1 U/ml GO, as indicated. EGFR was IPed from cell lysates, resolved by SDS-PAGE and IBed for total receptor, total tyrosine phosphorylation (p-EGFR) and specific Tyr-residue phosphorylation level (Y845, Y1068, Y1086, and Y1173). Protein aliquots of the cell lysates were also directly IBed for total and Y416 phosphorylated (active) c-Src (p-Src).

Article Snippet: PP1, a Src family inhibitor (Enzo Life Sciences), was dissolved in DMSO and added to the treatment medium at a final concentration of 5 µM.

Techniques: Incubation, SDS Page

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A549 cells were incubated (or not) with 5 µM PP1 for 45 min. and then treated (or not) for 15 min. with 100 ng/ml EGF or 30 min. 1 U/ml GO. A . EGFR was IPed from total cell lysates with the mAb 528 and IBed for Y416 phosphorylated c-Src (p-Src) and for total EGFR, as indicated. B . c-Src was IPed from total cell lysates and IBed for total c-Src and EGFR, as indicated.

Article Snippet: PP1, a Src family inhibitor (Enzo Life Sciences), was dissolved in DMSO and added to the treatment medium at a final concentration of 5 µM.

Techniques: Incubation