Fig. 3

Effect of increasing amounts of DI and full-length genome segment RNAs on expression of GFP from a segment 1 reporter gene construct. 293T cells were transfected with the segment 1-GFP plasmid, plasmids expressing PB1, PB2, PA and NP proteins, and increasing amounts of an additional PolI plasmid expressing a DI RNA (1/244, 1/244 KO, 2/265 or 3/262) or a full-length vRNA (segment 1, 4 or 6). Cells were examined for fluorescence 2 days after transfection. a. Pairs of cell monolayer images taken by phase-contrast (left) and epifluorescence microscopy (right). The amount of each plasmid expressing the various RNAs used as putative inhibitors is shown on the left. Control cells (top) were transfected with an empty vector (1 μg). b. Quantitation of fluorescence in cells generated in the presence of transfected plasmids expressing 1/244 DI, 1/244 KO DI, 3/262 DI, full-length segment 1 and full-length segment 6 RNAs, as indicated in the key. Columns show the mean of 3 (1/244 RNA, segment 1 and segment 6) or 2 (1/244 KO DI RNA and 3/262) independent experiments, and bars are standard errors of the mean. c Quantitation of fluorescence in cells generated in the presence of transfected plasmids expressing full-length segment 4 RNA (black columns) and DI 2/265 RNA (grey columns). Columns show the mean of 2 independent experiments, and bars are standard errors of the mean. Statistical significance determined using a two-tailed Student’s t test; p values for specific comparisons are shown