Journal: Scientific Reports

Article Title: A screen for novel hepatitis C virus RdRp inhibitor identifies a broad-spectrum antiviral compound

doi: 10.1038/s41598-017-04449-3

Figure Lengend Snippet: Assays with HCV genotype 3a replicon. ( A ) G418 resistant HCV-3a replicon expressing Huh7.5 cells were treated with indicated compounds for 48 h and the firefly luciferase activity was plotted as relative luciferase units (RLU). DMSO treated HCV-3a replicon expressing Huh 7.5 cells was taken as 100%. ( B ) The toxicity of these compounds in the replicon expressing cells was measured using WST-1 assay reagent. The values are depicted as percentages with the DMSO treated cells taken as 100%. ( C ) The replicon expressing cells were treated with varying concentrations of 66E2 and relative luciferase unit is plotted against the concentration of 66E2. EC 50 is the compound concentration that inhibits 50% of viral replication (RLU). ( D ) Huh7.5 cells were treated with indicated concentrations of 66E2 and cytotoxicity determined using WST-1 assay reagent. The values are plotted as percentages with the DMSO treated cells taken as 100%. CC 50 is the compound concentration that produces 50% of cytotoxicity. ( E ) Western blot analysis. Replicon expressing Huh7.5 cells were treated with different concentrations of 66E2 and CMC for 48 hours and expression of NS5A monitored using anti NS5A antibody. Expression of GAPDH was determined for loading control. ( F ) Reverse-transcriptase quantitative PCR analysis was performed to determine the levels of positive and negative sense HCV-3a RNA upon treatment with 66E2 and CMC at 5 μM for 48 h. The % mean is shown above the bars and the error bars are standard deviations. All the assays in the figure were performed in triplicates and results presented are representative of at least three independent assays.

Article Snippet: Antibodies against HCV NS5A (ab13833) and DENV (ab26837 and ab9202) were from Abcam and anti GAPDH was purchased from Santa Cruz Biotechnology (USA).

Techniques: Expressing, Luciferase, Activity Assay, WST-1 Assay, Concentration Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 1 RAC3 interaction with ERa is GTP and ligand dependent in vitro and in cells. (a) HIS-tagged RAC3, pulled down with Talon beads (Clontech), interacts with full-length ERa (Invitrogen) in a GTP and estradiol (E2) dependent manner. Western blot was probed with H222.2 and pan RAC antibody. (b) Quantification of western blot by LICOR software. Averaged integrated intensity of ERa bands was normalized to average integrated intensity of RAC3 bands. (c) RAC3 interacts with ERa in ligand dependent manner as measured by mammalian two- hybrid analysis of VP-16-ERa and GAL4 tagged RAC3 and RAC1 in MCF7 C4-12 cells. (d) Co-immunoprecipitation of ERa with RAC3 in MCF7 C4-12 þ Flag-ERa cells transfected with WT- RAC3. Western blot was probed with RAC and H222.2 antibodies and all bands were taken from the same blot. Empty wells were spliced out for clarity.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: In Vitro, Western Blot, Software, Immunoprecipitation, Transfection

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 2 RAC3 is an ERa co-activator. (a, d) RAC3 overexpression increases E2-induced ERE luciferase activity in MCF7 cells transfected with PGL2-ERE-luciferase, RTK and indicated constructs as described in Material and methods. MCF7 cells were treated with 10 nM E2, 10 nM E2 þ 1 mM ICI and ethanol for 6–24 h as indicated. (b, c) Western blots probed with anti-Myc antibody show comparable protein expression of Myc-tagged RAC3 and RAC1 or WT, T17 and V12 RAC3 mutants from an independent transfection. (e) RAC3 overexpression increases CCND1 gene expression in MCF7 cells transfected with RAC3 or vector. Gene expression was measured by TAQMAN assay. The symbol *P-value is 0.01. (f, g) RAC3 is present (f) and increases ERa occupancy (g) at the CCND1 promoter in MCF7 C4–12 cells stably transfected with Flag-ERa and transiently transfected with RAC3 or RAC1. Occupancy was determined by ChIP analysis and measured by real-time PCR as percent input. Western blot probed with anti-Myc antibody shows comparable protein expression of Myc-tagged RAC3 and RAC1 from an independent transfection.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Construct, Western Blot, Expressing, Gene Expression, Plasmid Preparation, TaqMan Assay, Stable Transfection, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 3 A single amino acid confers RAC3 co-activation of ERa. (a) Sequence alignment of RAC3 and RAC1. Boxed amino acids were mutated. (b, c) Mutations at S151 of RAC3 and RAC1 alter the ability of the GTPases to activate ERa-induced transcription as measured by ERE luciferase assay in MCF7 cells, which were transiently transfected with indicated constructs. (d) RAC3 S1515A mutation decreases the affinity of RAC3 for ERa in MCF7 C4-12 cells stably transfected with Flag-ERa and transiently transfected with RAC3 WT and RAC3 S151A. After a 1 h treatment with 10 nM E2, ERa was pulled down with H222.2 antibody. Western blot was probed with pan-RAC and ERa antibodies; all bands were taken from the same blot with empty wells omitted for clarity.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Activation Assay, Sequencing, Luciferase, Transfection, Construct, Mutagenesis, Stable Transfection, Western Blot

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 7 Clinical consequences of RAC3 expression in ERa positive human breast tumors. Gene expression data were obtained from the NKI patient database. (Chang et al., 2003; Chang et al., 2005) All analysis was done using Prism 5 for Mac OS X from GraphPad (La Jolla, CA, USA). (a, b) Increasing RAC3 expression decreases recurrence free survival shown in Kaplan–Meier curves of recurrence free survival in years. Increasing RAC1 expression had no effect. (c, d) Increasing RAC3 expression increases the proportion of patients with metastatic events. RAC1 expression levels had no statistically significant effect.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Expressing, Gene Expression

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 1. Temporal expression of A6L during VACV infection. (A) Schematic representation of vA6L-V5, a recombinant VACV en- coding A6 with a C-terminal V5 epitope tag. The relevant portion of the genome of vA6L-V5 with the inserted V5 tag (filled arrow) is depicted. For comparison, the corresponding portion of the WT WR genome is depicted with the positions of the open reading frames (open boxes with arrows) indicated by the nucleotide numbers in the complete WR genomic sequence (accession no. AY243312). (B) The kinetics of A6L expression. BS-C-1 cells were mock infected or in- fected with 10 PFU per cell of vA6L-V5. AraC was added to one of the infections at 40 g/ml to inhibit DNA replication. The cells were harvested at the indicated time (hours p.i. [hpi]) and analyzed by Western blotting (WB) with a MAb to V5 epitope as described pre- viously (15). The same amount of total proteins as determined by Bradford protein assay were analyzed.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Expressing, Infection, Recombinant, Comparison, Sequencing, Western Blot, Bradford Protein Assay

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 4. A6 protein expressed by vA6L-mut2 has a reduced stability at 40°C. (A) The A6 protein level in cells infected by vA6L-mut2 or vA6L-V5. BS-C-1 cells were infected with 10 PFU per cell of vA6L- mut2 or vA6L-V5 and maintained at 31 or 40°C. The cells were har- vested at 9 or 12 h p.i. and analyzed with SDS-PAGE and Western blotting (WB) with antibodies to the V5 epitope and VACV late protein D8. (B) Pulse-chase analysis of A6 protein. BS-C-1 cells were infected with vA6L-mut2 or vA6L-V5 at 40°C. At 12 h p.i., the cells were metabolically labeled with [35S]methionine-cysteine for 30 min. The cells were either harvested immediately (P) or replenished with normal medium and harvested 4 h later (C). The cell lysates as well as proteins precipitated from the cell lysates with anti-V5 antibody were analyzed with SDS-PAGE and autoradiography. IP, immunoprecipi- tate.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Infection, SDS Page, Western Blot, Pulse Chase, Metabolic Labelling, Labeling, Autoradiography

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 6. vA6L-mut2 is not defective for forming viral DNA-containing factories. (A). Localization of A6 protein in infected cells. HeLa cells grown on coverslips were infected with 0.1 PFU/cell of vA6L-V5 at 37°C. At 12 h p.i., the cells were fixed, permeabilized, blocked, and stained with a monoclonal antibody to V5 and a Cy2-conjugated secondary antibody. The DNA was stained with Hoechst dye. The images were acquired with an AX-70 Olympus fluorescence microscope. (B). BS-C-1 cells grown on coverslips were infected with 0.1 PFU/cell of vA6L-V5 or vA6L-mut2 at 40°C. At 12 h p.i., the cells were fixed, permeabilized, blocked, and stained with polyclonal antibody to VACV. The DNA was stained with Hoechst dye. The arrows point to typical DNA-containing viral factories.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Infection, Staining, Microscopy

Journal: British Journal of Pharmacology

Article Title: Role of H 2 O 2 in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II

doi: 10.1111/j.1476-5381.2012.01957.x

Figure Lengend Snippet: Earlier (day 10) effects of Ang II, with or without PEG-catalase, on renal Nox1 protein expression on day 10. Data are expressed as representative immunoblots with summary bar graphs (mean ± SEM values), part III, n= 4–7.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked for 1 h and incubated overnight at 4°C with specific antibody against Nox4 (sc-21860, 1:200, Santa Cruz Biotechnology or ET3174-1, 1:200, Epitomics, Burlingame, CA, USA), Nox1 (sc-5821, 1:200, Santa Cruz Biotechnology) or AT 1 receptor (sc-1173, 1:500, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot