Figure 5

Leptomycin B treatment does not alter cytoplasmic influenza PA, PB1, or PB2 mRNA in A549 cells. A549 cells were infected with influenza A and virus allowed to adhere for 1 hour at which time virus inoculum was removed and replaced with media containing 10nM leptomycin B (LMB) to inhibit Crm1-mediate nuclear export or untreated media as control. A. Media samples from mock infected, LMB treated and untreated infected cells (top rows infected at MOI 1.4, bottom row infected at 2.8 MOI) were collected 36 hours post infection and subject to HA assay using two-fold dilutions. B. Infected LMB treated and untreated cells were fractionated at 3.5 hours post infection. Cytoplasm and nuclear protein fractions were separated by SDS-PAGE and subject to Western blot to detect Nxf1 and Hsp90. C. RT-qPCR of RNA isolated from the cytoplasm fraction 3.5 hours post infection. RNA was quantified and equal concentrations subject to RT with oligo dT. Gene specific PCR was performed using primers to amplify PA, PB1, PB2, HA, and NP as indicated. Data shown is from one biological trial 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 0.05 as significant, no genes showed statistical difference in relative expression of RNA. PB2 p value was 0.105.