Figure 1.

The knockdown of HDAC4 expression promotes IAV infection. A–F, A549 cells were transfected with 1 nm control siRNA or HDAC4 siRNA for 72 h. The cells were then infected with IAV PR8 or CA09 strains at a m.o.i. of 1.0. After 24 h, the cells and the medium was harvested separately. A, total lysates of uninfected (UNI) and PR8-infected (INF) cells transfected with control siRNA (CT) or HDAC4 siRNA (HD4) were prepared, and the HDAC4 (140 kDa), PDI (57 kDa), and viral NP (56 kDa) polypeptides were detected by Western blotting. Note: the HDAC4 blot was reused to probe for PDI and NP, and the MW, UNI/CT, and UNI/HD4 lanes were combined with INF/CT and INF/HD4 lanes after cropping out the less relevant middle lanes of same blot. B, the HDAC4 and PDI bands in panel A were quantified using the Image Studio Lite software (LI-COR). The level of HDAC4 polypeptide in each sample was then normalized with corresponding PDI levels. Finally, the normalized level of HDAC4 polypeptide in control siRNA-transfected cells was considered 100% to compare its level in HDAC4 siRNA-transfected cells. C, A549 cells were transfected with either Lipofectamine (LF) alone or in complex with control siRNA or HDAC4 siRNA. After 72 h, the viability of cells was determined by MTT assay. Then, the viability of Lipofectamine only transfected cells was considered 100% to compare the viability of the cells transfected with control siRNA and HDAC4 siRNA. D, the culture medium from the cells transfected with control siRNA or HDAC4 siRNA and infected with IAV PR8 or CA09 strains was titrated on MDCK cells by microplaque assay. Then, the amount of viral progeny released from control siRNA-transfected cells was considered 1-fold to compare the amount of viral progeny released from HDAC4 siRNA-transfected cells. E, the total culture medium from the cells transfected with control siRNA or HDAC4 siRNA and infected with PR8 was concentrated by TCA precipitation, and the levels of viral HA (68 kDa) and viral NP were detected by Western blotting. Note: the HA blot was reused to probe for NP, and the MW lane was combined with CT and HD4 lanes after cropping out the less relevant middle lanes of same blot. F, total RNA from the cells transfected with control siRNA or HDAC4 siRNA and infected with PR8 or CA09 was isolated, and the levels of viral NP, viral M, and actin mRNAs were detected by qPCR. Then, the levels of NP and M mRNAs were normalized with the level of corresponding actin mRNA. Finally, the normalized levels of each viral mRNA in control siRNA-transfected cells were considered 1-fold to compare their levels in HDAC4 siRNA-transfected cells. G, the NP polypeptide level in infected cells in panel A was quantified and normalized as in panel B. Then, the normalized level of NP in control siRNA-transfected cells was considered 1-fold to compare its level in HDAC4 siRNA-transfected cells. H, A549 cells, transfected with control siRNA or HDAC4 siRNA for 72 h, were infected with PR8 at a m.o.i. of 0.1. After 6, 12, and 24 h of infection, the culture medium was titrated by microplaque assay to determine the titers of released viral progeny. Error bars represent the mean ± S.E. of three independent experiments (B, D, F, and G), biological replicates (H), or technical replicates (C). The asterisks represent p values mentioned in the text calculated by unpaired t test (B, F, and G) or ANOVA (C, D, and H), and indicate the significant differences in means. MW, molecular weight; CT, control siRNA; HD4, HDAC4 siRNA.