Study shRNA lentivirus vectors.

<p>^a NA—not available</p><p>Study shRNA lentivirus vectors.</p

Inhibition of HPV+ cervical cancer cells by lentiviral shRNAs that do not target human genes.

<p>Cell viability was assessed using a CellTiter Blue assay 5 days after infection. Each of the viruses was titrated for infection to determine its potency of inducing HeLa cell proliferation/viability inhibition. Percent cell viability (fluorescence) in the presence of lentiviral shRNAs was calculated by comparing with a control lentivirus without shRNA expression. Data represent the average ± standard deviation of 4 replicates from one of three independent experiments with similar results; (A) Inhibition of HeLa cell proliferation/viability; (B) Inhibition of proliferation/viability of CaSki cells.</p

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

<div><p>The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.</p></div

Loss of cell viability induced by PAK3 or SGK2 shRNAs.

<p>(A) HeLa cells were infected with PAK3 shRNA at a 1:10 dilution. Cell apoptosis was determined using a caspase 3/7 glo luciferase assay 72 hours after infection. The bar graph presents fold changes of caspase 3/7 activity (luminescence) induced by various shRNAs compared with a no-shRNA control. Data represent the average ± standard deviation of 4 replicates from one of two separate experiments with similar results; (B) Inhibition of proliferation/viability of HeLa cells by PAK3 shRNAs was assessed using a CellTiter blue assay 5 days after infection. The bar graph presents percent viability (fluorescence) of lentiviral shRNA-infected cells compared with a control lentivirus without shRNA expression. Data represent the mean ± standard deviation of three independent experiments; (C) SGK2 lentiviral shRNAs induced HeLa cell apoptosis. Data represent 4 replicates from one of two separate experiments with similar results; (D) SGK2 shRNAs inhibited proliferation/viability of HeLa cells. Data represent the average ± standard deviation of two independent experiments; (E) SGK2 lentiviral shRNAs induced autophagy of HeLa cells. Cells were infected with SGK2 lentiviral shRNAs at 1:16 and 1:32 dilution, respectively. 1 μM Rapamycin was included as an autophagy control on each plate. Cell plates were fixed 72 hour after infection and immunostained for induction of autophagy with a LC3B primary antibody, followed with an Alexa 488-conjugated secondary antibody. Images were captured using the confocal Opera High Content Imager. Images of SGK2 lentiviral shRNA-infected HeLa cells (~1200 cells/well) were captured and cell numbers counted. The average intensity of LC3B staining per cell was measured and calculated using a modified Capella (Perkin Elmer) algorithm. The bar graph presents the average ± standard deviation of LC3B staining intensity derived from two wells.</p

Rescue of SGK2 shRNA-induced phenotypes.

<p>(A) HeLa cells were transfected with serially diluted SGK2 expressing plasmids harboring silent mutations at the shRNA 2111 annealing site. Six hours after transfection, the cells were infected with SGK2 lentiviral shRNA 2111 (1:15 dilution). SGK2 mRNA expression levels were determined 72 hours after infection. The bar graph presents quantities of SGK2 mRNA normalized with GAPDH mRNA. Data represent the average ± standard deviation of 4 replicates from one of two experiments with similar results; (B) HeLa cell apoptosis induced by SGK2 shRNA 2111 was quantified with a caspase 3/7 glo assay 72 hours after infection. Two variants of SGK2 (alpha and beta) were used for the phenotype rescue analysis. Data represent the average ± standard deviation of two independent experiments; (C) HeLa cell proliferation/ viability inhibition induced by SGK2 shRNA 2111 was determined using the CellTiter Blue assay 5 days after infection. Data represents the average ± standard deviation of two independent experiments.</p

Knockdown of PAK3 and SGK2 by shRNAs.

<p>mRNA levels were determined by quantitative reverse transcription PCR analysis at 72 hours after infection with the respective shRNA vectors; control denotes infection with a vector encoding non-target shRNA. Each bar on the bar graph represents the average ± standard deviation of 4 replicates from one of three independent experiments with similar results. mRNA levels were normalized with GAPDH expression; PAK3 and SGK2 protein expression were determined 72 hours after infection using a Western immunoblot. GAPDH protein acted as a protein loading control for each sample. (A) PAK3 shRNA decreased PAK3 mRNA levels in HeLa cells; (B) PAK3 shRNAs reduced PAK3 mRNA expression in DMS-79 cells; (C) PAK3 shRNAs reduced PAK3 protein expression in DMS-79 cells. DMS-79 cell lysates were analyzed with PAK3 monoclonal antibody N-19; (D) SGK2 shRNAs reduced SGK2 mRNA levels in HeLa cells; (E) SGK2 shRNAs reduced SGK2 mRNA levels in GTL16 cells; (F) SGK2 shRNAs reduced SGK2 protein levels in GTL16 cells. GTL16 cell lysates were analyzed with SGK2 monoclonal antibody 3Q-2.</p

Impact of Pre-existing NS5A-L31 or -Y93H Minor Variants on Response Rates in Patients Infected with HCV Genotype-1b Treated with Daclatasvir/Asunaprevir

<p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>. </b><a href="https://link.springer.com/article/10.1007/s12325-016-0354-1">https://link.springer.com/article/10.1007/s12325-016-0354-1</a></p><p></p> <p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</p

Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

<div><p>Peginterferon lambda-1a (Lambda), a type III interferon (IFN), acts through a unique receptor complex with limited cellular expression outside the liver which may result in a differentiated tolerability profile compared to peginterferon alfa (alfa). In Phase 2b clinical studies, Lambda administered in combination with ribavirin (RBV) was efficacious in patients with hepatitis C virus (HCV) infection representing genotypes 1 through 4, and was associated with more rapid declines in HCV RNA compared to alfa plus RBV. To gain insights into potential mechanisms for this finding, we investigated the effects of HCV replication on IFN signaling in primary human hepatocytes (PHH) and in induced hepatocyte-like cells (iHLCs). HCV infection resulted in rapid down-regulation of the type I IFN-α receptor subunit 1 (IFNAR1) transcript in hepatocytes while the transcriptional level of the unique IFN-λ receptor subunit IL28RA was transiently increased. In line with this observation, IFN signaling was selectively impaired in infected cells upon stimulation with alfa but not in response to Lambda. Importantly, in contrast to alfa, Lambda was able to induce IFN-stimulated gene (ISG) expression in HCV-infected hepatocytes, reflecting the onset of innate responses. Moreover, global transcriptome analysis in hepatocytes indicated that Lambda stimulation prolonged the expression of various ISGs that are potentially beneficial to antiviral defense mechanisms. Collectively, these observed effects of HCV infection on IFN receptor expression and signaling within infected hepatocytes provide a possible explanation for the more pronounced early virologic responses observed in patients treated with Lambda compared to alfa.</p></div

Lambda induces broader and more sustained biological activities than alfa in naive hepatocytes.

<p>Microarray transcriptional profiling of naive iHLCs treated with 10 ng/mL of alfa or Lambda over the course of 48 hours. (A) Relative expression of 83 genes shown to be up-regulated >1.5-fold at all time points by one or both agents, normalized to untreated iHLC at the same time point. Genes indicated by dark blue overlay are consistently regulated by both agents throughout the time series, while genes indicated by light blue overlay show more sustained induction with Lambda compared to alfa. (B) Venn diagram displaying numbers of genes up-regulated (>1.5-fold change) following the indicated treatments. Significance values from MetaCore network analysis for the top six signaling pathways related to the 45 genes with sustained up-regulation by Lambda, and their corresponding <i>P</i> value for the list of 38 shared genes.</p

Impairment of alfa signaling in HCVcc-infected hepatocytes.

<p>(A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, <i>P</i> < 0.001.</p