Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Analysis of the expression of IFN-regulated genes in liver cells from pigs infected with HEV-3c and HEV-3f. Liver samples from 2 controls, 3 HEV-3c and 3 HEV-3f infected pigs were collected 8 days post-infection (peak excretion) and were analyzed using a RT 2 Profiler PCR array designed to study the expression of 84 genes involved in the IFN response. (A) Heat map showing the differential expression (fold regulation) of the 84 analyzed swine genes. Up- and down-regulated genes are colored in red and blue, respectively. (B) Graph representing the fold changes obtained for the different studied genes.

Article Snippet: First, we validated that HepaRG are able to respond to IFN-I using a PCR array (Human Type I Interferon Response RT2 Profiler PCR Array, Qiagen) to study the expression of 8 IFN and receptor genes and 76 genes involved in IFN signaling.

Techniques: Expressing, Infection, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Preliminary screen to analyze the expression of IFN-regulated genes in HepaRG cells infected with HEV-3f. HepaRG cells infected with HEV-3f at a MOI of 10 or 100 GE/cell and their matched non-infected controls were analyzed at different days (D) after infection using the Human Type I Interferon Response RT² Profiler PCR Array (Qiagen). The heat map shows the differential expression of 84 analyzed human genes. The color bar represents gene expression level where up- and down-regulated genes are colored in red and blue, respectively. For MOI 10, 1 sample per time point was analyzed. For MOI 100, 1 sample containing pooled RNA from triplicate samples was analyzed.

Article Snippet: First, we validated that HepaRG are able to respond to IFN-I using a PCR array (Human Type I Interferon Response RT2 Profiler PCR Array, Qiagen) to study the expression of 8 IFN and receptor genes and 76 genes involved in IFN signaling.

Techniques: Expressing, Infection, Quantitative Proteomics, Gene Expression

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Analysis of the expression of IFN-regulated genes in HepaRG cells infected with HEV-3f. HepaRG cells infected with HEV-3f at a MOI of 100 GE/ml for 100 days and their matched non-infected controls were analyzed using RT 2 Profiler PCR arrays designed to study the expression of 168 genes involved in the IFN response. (A) Heat map showing the differential expression (fold regulation) of the 168 analyzed human genes. Up- and down-regulated genes are colored in red and blue, respectively. Fold regulations were calculated by the RT 2 Profiler PCR Arrays & Assays Data Analysis software (Qiagen) using average ΔCT values obtained from 2 independent experiments performed in triplicates. (B) Graph representing the fold changes obtained for the different studied genes. Fold changes with a p-value ≥ 0.05 are indicated by an asterisk (*).

Article Snippet: First, we validated that HepaRG are able to respond to IFN-I using a PCR array (Human Type I Interferon Response RT2 Profiler PCR Array, Qiagen) to study the expression of 8 IFN and receptor genes and 76 genes involved in IFN signaling.

Techniques: Expressing, Infection, Quantitative Proteomics, Software

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Validation of the PCR array data. (A) HepaRG cells were infected or not with HEV-3f for 100 days at MOI 100 and the expression of selected genes shown by PCR array to be up-regulated or unchanged was analyzed by RT-qPCR. The results shown are the geometric means ± SD from 3 to 4 replicate samples and are representative of 2 independent experiments. GUSB , B2M and GAPDH were used as reference genes. Unpaired t-test with Welch’s correction Mann-Whitney test, *: p<0.05 (B) Immunoblot showing the expression of the protein encoded by IFIH1 , DDX58 and IRF1 in HepaRG cells infected with HEV-3f or not (NI) for 100 days at MOI 100 for 3 replicate samples. Representative blots from 2 independent experiments are shown. ORF2 and actin protein levels were also detected as control of infection and loading, respectively. (C) Detection of CXCL10 in the supernatant of non-infected HepaRG (NI) or HepaRG cells infected with HEV-3f for 100 days at MOI 100 (GE/cell) by ELISA. The results shown are the means ± SD from 4 replicate samples and are representative of 2 independent experiments. Unpaired t-test with Welch’s correction, *: p<0.05.

Article Snippet: First, we validated that HepaRG are able to respond to IFN-I using a PCR array (Human Type I Interferon Response RT2 Profiler PCR Array, Qiagen) to study the expression of 8 IFN and receptor genes and 76 genes involved in IFN signaling.

Techniques: Biomarker Discovery, Infection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Barrier-to-autointegration factor 1 promotes gammaherpesvirus reactivation from latency

doi: 10.1038/s41467-023-35898-2

Figure Lengend Snippet: iSLK.219 cells were transfected with NTC siRNA or BANF1 targeting siRNA for 48 h prior to the addition of 25 ng/mL doxycycline. TREx-BCBL1-RTA cells were transduced with lentiviral shRNA for 72 h prior to the addition of 1000 ng/mL doxycycline. A iSLK.219 cell lysates were collected at 72 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B iSLK.219 cell lysates were collected at 48 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. C iSLK.219 culture supernatant was harvested at 72 h post-doxycycline treatment and analyzed by IFNβ ELISA. D Cells were harvested for RNA at 48 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and RT-qPCR was subsequently performed to determine ISG mRNA expression levels. E iSLK.219 cDNA as prepared in ( D ) was analyzed by Human Type I Interferon Response RT 2 Profiler PCR Array (Qiagen, GeneGlobe ID: PAHS-016Z). Data shown are the Z-score of the fold change (2 -ΔΔCt ) over the geometric mean expression of four housekeeping genes averaged over two independent biological replicates. The heatmap was prepared using Partek Flow. F Culture supernatants were harvested at either 72 h (iSLK.219) or 96 h (TREx-BCBL1-RTA) post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. G iSLK.219 culture supernatants were transferred to naive HEK293 cells at 72 h post-doxycycline treatment. GFP + infected cells were measured at 48 h post-infection by fluorescent microscopy and ( H ) quantified by flow cytometry. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.

Article Snippet: E iSLK.219 cDNA as prepared in ( D ) was analyzed by Human Type I Interferon Response RT 2 Profiler PCR Array (Qiagen, GeneGlobe ID: PAHS-016Z).

Techniques: Transfection, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Expressing, DNA Extraction, Infection, Microscopy, Flow Cytometry, Two Tailed Test