Journal: Nucleic Acids Research

Article Title: Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex

doi: 10.1093/nar/gkad483

Figure Lengend Snippet: Production of m7GTP in Nsp14-expressing cells. ( A ) Detection of m7GTP in extracts prepared from cells cultured under the indicated conditions using CE-MS. Each analysis was performed in triplicate. Peak migration times are shown in parentheses. ( B ) 293F_Nsp14_wt2 cells were transiently transfected with the FLAG-tagged DcpS expression vector. Twenty-four hours after transfection, 2.5 μg/ml DOX was added to the culture medium, and the cells were cultured for an additional 48 h. The cells were fixed and subjected to IFA using anti-FLAG M2 mAb followed by FISH using a Cy3-labeled oligo-dT 50 probe. The cells were observed by confocal microscopy. Maximum intensity projections of a single stack (10 consecutive slices, 0.35 μm z -distance) of images are shown. In the merged picture, the fluorescent signals of FLAG-DcpS and poly(A) + RNAs were pseudocolored blue and red, respectively. ( C , D ) 293F_hACE2_DcpS_29 cells were left untreated (–) or induced by DOX for 24 h (DcpS). The cells were mock transfected (mock) or transfected with the GFP-Nsp14 expression vector (Nsp14) and cultured for an additional 48 h. (C) Whole-cell extracts were subjected to Western blotting using the indicated antibodies. (D) Total RNA was subjected to qRT-PCR analysis as in Figure . The amount of downstream RNA was normalized to that of CDS RNA. The data are presented as the means ± SDs of three biological replicates. * means P value < 0.05.

Article Snippet: The cells were lysed in RIPA buffer, and the soluble fractions were adjusted to final concentrations of 1% SDS and 0.5 M LiCl, then heat denatured and subjected to oligo-dT cellulose (Wako pure chemicals) chromatography.

Techniques: Expressing, Cell Culture, Migration, Transfection, Plasmid Preparation, Labeling, Confocal Microscopy, Western Blot, Quantitative RT-PCR