a) Confocal microscopy indicates decreased NP translocation to nuclear on Hsp40 knockdown. A549 cells were transfected with pooled siRNA against Hsp40/DnaJB1 and control non-targeting siRNA. 24 h post-transfection, cells were infected with X-31 virus at MOI = 5 in the presence of cycloheximide. Cells were fixed 4 h post-infection followed by immunostaining for NP (red) and Hsp40 (green). (b) Quantitative analysis of nucleoprotein localization in non-targeting siRNA treated and Hsp40 siRNA treated −CHX and +CHX cells. The images were acquired under confocal microscope with 40X objective lens and percentage of cells with nuclear localized NP among total cells was calculated. The results are mean and standard deviation of four replicates. (c,d) Western blot analysis further confirms the effect of Hsp40 knockdown on NP and PB2 localization. From the similar experiment as shown in Fig. 4a, cells were harvested 4 h post-infection and subjected to cellular fractionation followed by western blotting. HDAC1 and GAPDH were used as loading control for nuclear fraction and cytoplasmic fraction respectively. Densitometry analysis was performed using ImageJ. NP, PB2 and Hsp40 levels in nuclear fraction were normalized with HDAC1 levels and relative nuclear protein levels in siRNA treated cells with reference to control siRNA treated cells were calculated and plotted. 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.