26 Compared with control treatments, cyclopamine reduced cell-ass

26 Compared with control treatments, cyclopamine reduced cell-associated HCV RNA by 70%, mirroring the observed decreases in Shh and Gli1 expression (Fig. 2A). Cyclopamine treatment also noticeably reduced the extent of infection as ascertained by immunofluorescence using antibody to the viral Core protein (Fig. 2B). Reductions in HCV Core content correlated strongly with the drop in Shh expression that followed cyclopamine treatment. To further characterize the effect Selleckchem Tanespimycin of cyclopamine on infected cells, we performed a time course experiment in which we isolated RNA from JFH1 infected Huh7.5 cells treated with cyclopamine and controls at 24, 48, and 72 hours postinfection

(Supporting Fig. 4). HCV RNA mirrored reductions in Gli1 RNA beginning at 24 hours and maximizing by 48-72 hours. In order to ascertain whether cyclopamine treatment was associated with changes in cell viability, we performed an analysis of LDH levels in supernatant media under different conditions. LDH levels were comparable between uninfected selleck chemicals llc and infected cells regardless of treatment (Supporting Fig. 5), indicating that reduced HCV replication was not due to potential toxic effects of cyclopamine treatment. Finally, we performed a FFU assay to quantify infectious virus from supernatant media at 72 hours from infected cells after cyclopamine and control treatment (Supporting Fig. 6).

Cyclopamine treatment led to a one log

reduction in focus forming units/mL compared with control treated cells. To further verify these results obtained with pharmacologic inhibition, we also used a neutralizing antibody to Shh (5E1) to inhibit pathway activity in Huh7.5 cells and observed similar reductions in HCV RNA, Shh, 上海皓元医药股份有限公司 and Gli1 observed with chemical inhibition (Fig. 2C). We next examined if recombinant N-terminal fragments of Shh, an agonist of the Hh pathway, would promote HCV viral titers in Huh7 cells. Incubation with exogenous Shh for 48 hours produced increased Shh and Gli1 transcripts and caused a 2-fold increase in HCV RNA levels (Fig. 3). It should be noted that JFH1 infection alone produced increased Shh and Gli1 transcripts and protein, and the increase in Shh expression paralleled the increase in core protein over time postinfection (Supporting Fig. 7).22 We replicated the increase in Huh7 permissiveness results using SAG, an Hh agonist that acts downstream by directly binding to Smoothened. SAG treatment resulted in a 3-fold increase in Hh pathway transcripts, and a corresponding 3-fold increase in HCV RNA levels (Fig. 4A). Corresponding increases in protein expression levels were observed (Fig. 4B). To confirm that this increase in HCV RNA correlated with functional virus, we used supernatants collected from the above experiment to infect naïve Huh7 cells.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>