Journal: bioRxiv

Article Title: Delta glutamate receptor conductance drives excitation of dorsal raphe neurons

doi: 10.1101/855353

Figure Lengend Snippet: A graphic representation of AAV vectors used in conjunction with AAV-Cas9 to encode eGFP reporter only (top, AAV-empty) or mouse Grid1 guide RNA with eGFP reporter (bottom, AAV-GRID1).

Article Snippet: The AAV packaging plasmid encoding a nuclear envelope-embedded eGFP reporter (Addgene 131682) was constructed by amplifying the KASH domain from (Addgene 60231, a gift of Feng Zhang) and fusing it (in-frame) to the end of coding region for eGFP in (Addgene 60058, pOTTC407) using ligation-independent cloning (AAV-empty, ). gRNA was cloned into a mU6 expression cassette and then moved into an AAV backbone expressing a nuclear envelope-embedded (KASH-tagged) eGFP reporter (Addgene 131683) by PCR amplification and ligation-independent cloning (AAV-GRID1).

Techniques:

Journal: Science translational medicine

Article Title: Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection

doi: 10.1126/scitranslmed.3010286

Figure Lengend Snippet: (A) Human hepatocytes (Huh7.5.1 cells) were infected with wild-type HCVcc in the presence of the compounds at 10 μM overnight followed by incubation with compound treatment for an additional 48 hours. Viral RNA was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). (B) Primary human hepatocytes were infected with wild-type HCVcc in the presence of (S)-CCZ titration overnight followed by incubation with (S)-CCZ titration for an additional 48 hours. Intracellular viral RNA levels were evaluated by qRTPCR. (C) In the presence of compound treatment, Huh7.5.1 cells were passaged every 3 days for seven passages and were plated on 96-well plates 3 days before ATPlite assay to measure cell viability. (D) HCV replication cycle assays were carried out with (S)-CCZ at 10 μM. Cyclosporin A (10 μM) was a control in HCV single-cycle infection (HCVsc), transient replicon genotype 1a, and replicon genotype 1b and 2a assays. Bafilomycin A1 (10 nM) was used as a control in HCV pseudoparticle (HCVpp) genotype 1a and 1b, vesicular stomatitis virus G pseudoparticle (VSV-Gpp), and murine leukemia virus pseudoparticle (MLVpp) assays. Results were normalized to DMSO. RLU, relative luminescence units. GT, genotype. (E) At t = −2 hours, HCV-Luc was incubated with Huh7.5.1 cells at 4°C for 2 hours for attachment. At t =0 hours, the unbound virus was removed, and the plates were moved to 37°C to allow synchronous infection and incubated for 48 hours before virus load measurement. (S)-CCZ (10 μM), bafilomycin A1 (10 nM), and sofosbuvir (10 μM) were added either continuously or at the indicated time points and incubated for 2 hours. (F) Results from (E) were normalized to DMSO continuous treatment. Data are means of replicates ± SEM (n ≥ 3). *P < 0.05, **P < 0.005, ***P < 0.0001, versus DMSO (Student’s t test).

Article Snippet: Plasmids encoding HCV genotype 1a envelope proteins were provided by S. Ray (Johns Hopkins University).

Techniques: Infection, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Titration, Control, Virus