MeTAFlow assay of HEK293T cells expressing Nsp1. (A) Schematic representation of MeTAFlow. Briefly, OPP molecules are incorporated into growing peptide chains then fluorescently labeled via CuAAC reaction. mRNA molecules are labeled with fluorescent, poly(A)-targeting molecular beacons (MB-oligo[dT]). Simultaneous measurement of nascent protein and mRNA abundance via their fluorescent signals in single cells is detected with flow cytometry. (B) MeTAFlow analysis of untransfected (unt), (C) Nsp2-transfected (Nsp2), and (D) Nsp1-transfected (Nsp1) HEK293T cells. The populations in panels B–D are OPP-AF488+/MB-Cy5+ cells. Gates show a population of unchanged cells (P1), as established by the baseline untransfected cells, and a second population of cells (P2) with reduced nascent protein and mRNA abundance. The Pearson correlation coefficient (r) for the nascent protein levels to the total mRNA abundance is given for each condition. (E) Comparison of Nsp1 expression levels in the Nsp1-transfected cells’ unchanged population (P1, gray) and population with reduced parameters (P2, purple) using Strep-Tactin XT conjugated to DY549 that detects Strep-Tag II fused to Nsp1. (F) Nascent polypeptide levels as measured by OPP fluorescence for P1 (gray) and P2 (purple) of Nsp1-expressing cells. (G) Measurements of total mRNA abundance by molecular beacon signal for P1 (gray) and P2 (purple) of Nsp1-expressing cells. The populations in panels E–G are OPP-AF488+/MB-Cy5+ cells. Data shown are representative examples of experiments performed in triplicate.
