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
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