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

Impact of Arf-GEF depletion on the cis-Golgi morphology.

<p>Huh-7 cells were transfected with siRNA targeting GBF1, BIG1, BIG2, or both BIG1 and BIG2. Cells were fixed 3 days after transfection and processed for the immunofluorescent detection of GM130 (shown in red). The nuclei were stained with DAPI (shown in blue). Representative confocal images are presented. Bar, 20 µm.</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

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

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

Selection of BFA-resistant cell lines.

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

HCV infection in BFA-resistant cells.

<p>Cells of the indicated cell lines were infected for 2<i>Renilla</i> luciferase in the presence of the indicated concentrations of BFA. The virus was removed and the cells were left in the presence of BFA for another 6-h period. Cells were lysed in <i>Renilla</i> lysis buffer at 24 hpi, and the luciferase activity was quantified as a measure of HCV infection. Results were expressed as percentages of the values obtained with no BFA. Error bars represent the SD of 3 experiments performed in triplicates. +, values below 0.1%.</p

Serum albumin and apolipoprotein E secretion in BFA-resistant cells.

<p>(A) Albumin secretion. Cells of the indicated cell lines 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 4 independent experiments performed in duplicates. (B) Basal HSA expression levels. HSA of the indicated cell lines were quantified from cell lysates (in the absence of BFA treatment) by ELISA and normalized to the total protein concentration. (C) Apolipoprotein E (apoE) secretion. Cells were cultured in 24-well plates, in the presence of the indicated concentrations of BFA for 8 h. The amounts of apoE in cell lysates and culture media and of tubulin in cell lysates were analyzed by immunoblotting. Error bars represent the SEM of 3 independent experiments.</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