primary human lung mrc 5 fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: Primary lung fibroblasts (MRC-5) were infected with WT or F412V at an MOI of 1. At 2 hpi, virus was washed out and media were added containing either 0.1% DMSO or 0.1% DMSO with 1.6 µM GCV. At 96 hpi, cells were harvested and total DNA was isolated for Illumina sequencing. After sequence alignment and removal of reads mapping to the human genome, viral reads were quantified and plotted for each condition. (A and B) Representative sequence coverage plots of total unique (dark blue) viral reads from WT (A) and F412V (B) infected cells treated with DMSO vehicle (top) or GCV in vehicle (bottom). The red dotted line represents the location of the oriLyt (location ∼140,000bp in the genome). Top (cyan) and bottom (purple) strand reads are depicted separately. Only unique reads that map to only one location in the reference genome were counted. (C) Total reads per million (top) and reads distant from oriLyt (bottom) were quantified. Non-parametric Wilcoxon testing was used to determine statistical significance. The resulting p-values were adjusted using the Benjamini-Hochberg method: **, p<0.01; ns, not significant) (D) Sequence coverage decay plots representing the normalized coverage at the oriLyt location up to 50kb moving away from the oriLyt in both directions. (E) Distribution of decay slopes and pairwise comparisons of adjusted p-values of WT and F412V treated with either DMSO or GCV. The slopes estimated per sample were compared across groups using pairwise permutation testing (with BH correction) to assess significant differences in replication bias patterns between conditions: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Virus, Isolation, Illumina Sequencing, Sequencing

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: Growth-arrested MRC-5 fibroblasts were transduced with lentiviruses to express shRNAs targeting Luc or three Y-family polymerases: polη, polι, or polκ in combination (A-C) or individually (D and E). (A) Following two days of selection by puromycin (2 µg/mL), cells were lysed and RNA was harvested for analysis by RT-qPCR. Knockdown efficiency was determined by ΔΔCt analysis between Luc and experimental conditions with cellular H6PD as a housekeeping control. Statistical significance was determined by two-tailed, paired t -test with Bonferroni correction. Asterisks (* P <0.05, ** P < 0.01) represent statistically significant differences relative to Luc. (B) Transduced fibroblasts were infected at an MOI of 1 with WT or F412V. At 2 hpi, virus was washed out and media was replaced containing various concentrations of GCV. At four days post infection, cells and supernatant were collected for analysis by TCID 50 . For each condition, virus yield at each GCV concentration was compared to the DMSO control and plotted as percent yield reduction for three biological replicate experiments. Curves were similarly fitted for WT and F412V in each individual experiment to obtain ED 50 values and CIs for each condition. (C) The ED 50 values from three biological replicate experiments were used for a ratio paired t test comparing viruses within each condition. ED 50 mean values are plotted with standard deviation error bars. (D-E) Transduced fibroblasts were infected with (D) WT or (E) F412V at an MOI of 1. At 2 hpi, virus was washed out and media was replaced containing either 0.1% DMSO or 0.1% DMSO plus 1.6 µM GCV, and at 4 days post infection, cells and supernatant were collected for analysis by TCID 50 . Percent virus yield in GCV-treated conditions compared to the DMSO control in each condition was plotted for five biological replicate experiments. Statistical significance was determined by a ratio paired t test: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Transduction, Selection, Quantitative RT-PCR, Knockdown, Control, Two Tailed Test, Infection, Virus, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

doi: 10.64898/2026.05.21.726832

Figure Lengend Snippet: (A) MRC-5 fibroblasts were infected at an MOI of 1 with WT or F412V. At 2 hpi, virus was washed out and media was replaced containing various concentrations of CDV. At four days post infection, cells and supernatant were collected for analysis by TCID 50 . For each condition, virus yield at each CDV concentration was compared to the DMSO control and plotted as percent yield reduction for three biological replicate experiments. Curves were similarly fitted for WT and F412V in each individual experiment to obtain ED 50 values and CIs for each condition. (B-C) Growth-arrested MRC-5 fibroblasts were transduced with lentiviruses to express shRNAs targeting Luc or polκ. These fibroblasts were infected with (B) WT or (C) F412V at an MOI of 1. At 2 hpi, virus was washed out and media was replaced containing either 0.1% DMSO or 0.1% DMSO plus 2.4 µM GCV, and at 4 days post infection, cells and supernatant were collected for analysis by TCID 50 . Percent virus yield in CDV-treated conditions compared to the DMSO control in each condition was plotted for three biological replicate experiments. Statistical significance was determined by a ratio paired t test: *, p<0.05; ns, not significant.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Virus, Concentration Assay, Control, Transduction

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of CoV-induced ROs and Cer in lung-derived cells. (A) Adenocarcinoma cell line A549-ACE2 (for SARS-CoV-2) or A549-CD13 (for HCoV-229E) or (B) primary lung fibroblasts MRC-5 cells (for HCoV-229E and MERS-CoV) were infected with an MOI of 3 for 8 hpi. The fixed cells were then permeabilized with 0.5% saponin and stained against dsRNA (red) and Cer (green). DAPI was used for staining of nuclei. Colocalization signals, rates and MCCs were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Derivative Assay, Infection, Staining