Figure 4.

The caspase 3 cleaves HDAC4 polypeptide in infected cells. A, MDCK cells were infected with PR8 at a m.o.i. of 0.5 and, after removing the virus inoculum treated with NH₄Cl (10 mm), MG132 (20 μm), or caspase 3 inhibitor (Cas3-I; 40 μm). After 24 h, the uninfected (UNI) and infected (INF) cells were harvested, total cell lysates were prepared, and the HDAC4, PDI, viral NP, and full-length caspase 3 (Cas3-FL) and cleaved caspase 3 (Cas3-CL) polypeptides were detected by Western blotting. Note: the HDAC4 blot was reused to probe for PDI and NP. Arrows indicate the HDAC4 polypeptide cleavage products. B, the full-length HDAC4 polypeptide level in panel A was quantified and normalized as described in the legend to Fig. 1B. Then, the normalized level of HDAC4 polypeptide in mock-treated uninfected (UNI) cells was considered 100% to compare its levels in mock-, NH₄Cl-, MG132-, and Cas3-I-treated infected (INF) cells. C, A549 cells were transfected in duplicate with 10 nm control siRNA (CT) or caspase 3 siRNA (Cas3) for 72 h. One set of cells was infected with PR8 at a m.o.i. of 1.0. After 24 h, cells were harvested and the HDAC4, NP, and actin polypeptides were detected in total cell lysates by Western blotting. Note: the HDAC4 blot was reused to probe for actin and NP. Arrows indicate the HDAC4 polypeptide cleavage products in the higher exposure of the dotted rectangle. D, the full-length HDAC4 polypeptide level in panel C was quantified and normalized as described in the legend to Fig. 1B. Then, the normalized level of HDAC4 polypeptide in uninfected/control siRNA-transfected cells (UNI/CT) was considered 100% to compare its levels in infected/control siRNA-transfected cells (INF/CT) and infected/caspase 3 siRNA-transfected cells (INF/Cas3). E, the second set of cells from panel C was processed to detect the levels of caspase 3 and actin mRNAs by qPCR. Then, the levels of caspase 3 mRNA in control siRNA-transfected cells and caspase 3 siRNA-transfected cells were normalized with corresponding actin mRNA levels. Finally, the normalized level of caspase 3 mRNA in control siRNA-transfected cells (CT) was considered 100% to compare its level in caspase 3 siRNA-transfected cells (Cas3). Error bars represent the mean ± S.E. of three independent experiments. The asterisks represent p values mentioned in the text calculated by ANOVA (B and D) or unpaired t test (E), and indicate the significant differences in means. MW, molecular weight.