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. 2016 Jan 11;6:19063. doi: 10.1038/srep19063

Figure 2. Drug mediated inhibition of Hsp impedes nuclear import of incoming IAV NP in virus infected cells.

Figure 2

(a) Confocal analysis indicates diminished nuclear accumulation of NP after KNK437 treatment. A549 cells were pretreated with DMSO or KNK437 for 6 h. Cells were infected with PR8 virus at MOI = 5 and treated with DMSO, KNK437 or nucleozin in the presence of cycloheximide (CHX). Cells were fixed at 4 h p.i. and observed under confocal microscope with 60X. Cells were stained using anti-NP and anti-Hsp40 primary antibody, followed by Alexa 594 and Alexa 488 conjugated secondary antibodies respectively. (b) Quantitative analysis of nucleoprotein localization in mock infected, DMSO, KNK437 and nucleozin treated −CHX and +CHX cells. The images acquired under confocal microscope at low magnification were used to calculate the percentage of cells with nuclear localized NP among total cell population. The results are mean and standard deviation of four replicates. (c,d) Western blot analysis shows decrease in NP levels in nuclear fraction after KNK437 treatment. A549 cells were pretreated with DMSO or KNK437 for 6 h. Cells were infected with X-31 virus at MOI = 5 and treated with DMSO, KNK437 or nucleozin. Cells were harvested 4 h post-infection followed by cellular fractionation and western blotting. Densitometry analysis was performed using ImageJ. NP and Hsp40 levels were normalized with HDAC1 levels and relative nuclear protein levels in KNK437 and nucleozin treated cells with reference to DMSO treated cells were calculated and plotted. (e) Nuclear to cytoplasmic ratio of NP protein levels decreases significantly in KNK437 treated cells. Relative NP protein levels in different fractions were calculated by densitometry analysis of western blots after cellular fractionation. Protein levels obtained were normalized by the total protein amount loaded as quantitated by Bradford assay. Data show mean ± S.D. from one representative experiment (n = 3) of at least three independent experiments. Statistical significance was determined using Student’s t test. *p < 0.05.