BFA sensitivity of the cis-Golgi of Huh-7, R1, R2 and MDCK cells.

<p>Cells were treated for 30 minutes with increasing concentrations of BFA, fixed and processed for the immunofluorescent detection of GM130. For each condition, approximately 100 cells were scored for their cis-Golgi morphology, as either intact or scattered. For each cell line, the percentages of cells with intact cis-Golgi morphology were plotted against BFA concentrations.</p

Immunoblot analysis of GBF1 and Arf1 expression in R1 and R2 cells.

<p>Equal amounts of lysates of the indicated cell lines were analyzed by immunoblotting for the expression of GBF1, Arf1 and β-tubulin.</p

Half maximal effective concentrations of BFA effects on the cis-Golgi morphology and HSA secretion.

<p>Half maximal effective concentrations of BFA effects on the cis-Golgi morphology and HSA secretion.</p

Mutation detected in GBF1 of R1 and R2 cells.

<p>(A) A fraction of the electrophoregrams corresponding to the sequence of GBF1 from the indicated cell lines is presented. The nucleotide and amino-acid sequences are indicated. The position of the mutation is indicated by an arrow. (B) Huh-7 cells were transfected with expression plasmids for GBF1-M832L, GBF1 inactive mutant E794K, or YFP. Transfected cells were submitted to a cell viability assay, as explained in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074491#pone-0074491-g001" target="_blank">figure 1</a>. Results were expressed as percentages of the values obtained with no BFA. Error bars represent the SEM of 3 independent experiments performed in triplicates. (C) Transfected cells were seeded in 12-well plates, and cultured in the presence of BFA for 24 h. The amounts of human serum albumin (HSA) in the conditioned culture media and in cell lysates were quantified with an ELISA assay and expressed as percentages of HSA secretion. Error bars represent the SEM of 3 independent experiments performed in duplicates.</p

Viability of BFA-resistant cells.

<p>Sub-confluent cells of the indicated cell lines were cultured in 96-well plates in the presence of the indicated concentrations of BFA or of 0.2% ethanol (BFA stock solvent) for 24 h. Viability was assessed using an MTS assay. The absorbance of the ethanol-treated sample is expressed as 100%. Results were expressed as percentages of the values obtained with no BFA. Error bars represent the SEM of 2 independent experiments performed in triplicates.</p

Selection of BFA-resistant cell lines.

<p>Selection of BFA-resistant cell lines.</p

Morphology of BFA-sensitive compartments of R1 and R2 cells.

<p>Cells of the indicated cell lines were treated with 2.5 µg/ml (A) or 5 µg/ml (B, C) BFA for 30 minutes, fixed and processed for the immunofluorescent detection of GM130 (A), TGN46 (B), or the transferrin receptor (C). The nuclei were stained with DAPI. Representative confocal images are presented. Bar, 20 µm.</p

Impact of GBF1 and Arf1 depletion on HCV replication in R1 and R2 cells.

<p>(A) Cells of the indicated cell lines were transfected with siRNA targeting GBF1, BIG1, or PI4KIIIα (PI4KA), and infected with HCVcc. Cells were lysed at 30 hpi and the expression of NS5A, GBF1, and β-tubulin were analyzed by immunoblotting. (B) For each cell line, NS5A expression is expressed as a percentage of the values obtained for mock-transfected cells. (C) Cells of the indicated cell lines were transfected with siRNA targeting Arf1 or PI4KIIIα (PI4KA), or with a non-targeting control siRNA, and infected with HCVcc. Cells were lysed at 30 hpi and the expression of NS5A, Arf1, and β-tubulin were analyzed by immunoblotting. (D) For each cell line, NS5A expression is expressed as a percentage of the values obtained for the control siRNA. Error bars represent the SD of 3 independent experiments. +, NS5A below the detection limit.</p

Mutations found in the coding sequence of GBF1 in Huh-7 cells.

a<p>Genbank NM_001199379.1.</p

Data_Sheet_1_A reporter cell line for the automated quantification of SARS-CoV-2 infection in living cells.PDF

The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose–response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2.</p