Figure 1

Expression of dominant negative Nxf1 decreases virus production and results in cytoplasmic reduction of select influenza mRNAs. A549 cells were transfected with plasmid to express dominant negative Nxf1 (DN) or vector control (vec) and infected with influenza A Udorn at 2.5 MOI 48 hours post transfection. A. Virus production at 12 hours post infection with influenza A Udorn. An asterisk indicates statistical difference between cells transfected with DN-Nxf1 compared to vec-control. Data presented is from biological triplicate trials. B. Cells transfected with DN-Nxf1 or vector for 48 hours and subsequently infected with influenza A Udorn at 2.5 MOI for 3.5 hours were fractionated. Cytoplasm and nuclear protein fractions were separated by SDS-PAGE and subject to Western blot to detect SP1 and Hsp90. Shown is a representative blot from one biological trial. C. RT-semi-qPCR of RNA isolated 3.5 hours post infection from the cytoplasm fraction. RNA was quantified and equal concentrations subject to RT with oligo dT. Gene specific PCR was performed using primers to amplify NP, PA, PB1 and PB2 as indicated. Data show sequential PCR cycles from three biological independent trials. D. RT-qPCR of RNA isolated from one of the above biological trials performed in triplicate. Delta Ct was calculated to determine relative RNA expression. Raw CT values were analyzed in Microsoft Excel using 2^{ΔCt(average control- average treated)}. Standard error was obtained by calculating the standard deviation of the sample set divided by the square root of the sample set size, and indicated using error bars. Significance was determined using a two-tailed T-Test conducted in Microsoft Excel, and judging any p value less than .05 as significant, indicated by an asterisk.