primary human lung mrc 5 fibroblasts  (ATCC)

Journal: Nucleic Acids Research

Article Title: Human cytomegalovirus regulates host DNA repair machinery for viral genome integrity

doi: 10.1093/nar/gkaf1445

Figure Lengend Snippet: USP1 restricts CMV replication. MRC-5 fibroblasts were reverse-transfected with a siRNA pool targeting UAF1 or USP1 or an NTC. At two days post-transfection, cells were infected with CMV TB40/E-GFP wild-type (WT) or TB40/E-∆ UL138 STOP at an MOI of 1 and cells were harvested at 96 hpi. ( A ) Viral genome copy number was determined by qPCR using a TB40/E BAC standard curve and primer set designed for the region of the viral genome encoding the β2.7 transcript. The graph represents the fold change in viral genome copy number relative to siNTC. ( B ) Virus yields were measured by TCID 50 and normalized relative to the WT siNTC condition. For panels (A) and (B), statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. Asterisks (** P < .01, *** P < .001, **** P < .0001) represent statistically significant differences determined for four independent experiments. ns, nonsignificant. ( C ) Whole cell lysates were collected at two days post-transfection to determine efficiency of USP1 knockdown by immunoblotting using monoclonal antibodies for USP1 and tubulin as a loading control and secondary antibodies conjugated to DyLight™ 680 (mouse) or 800 (rabbit). Densitometry analysis of three biological replicates reveals an average 68% knockdown compared to siNTC. ( D ) At two days post-transfection, RNA was collected for RT-qPCR to determine the efficiency of UAF1 knockdown relative to siNTC. H6PD was used as a housekeeping control for cell number. Over all experiments, an average knockdown efficiency of 81% was achieved. A paired t- test was used to determine statistical significance, **** P < .0001, over multiple independent experiments.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 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: Transfection, Infection, Virus, Knockdown, Western Blot, Bioprocessing, Control, Quantitative RT-PCR

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