FIG. 4.

Knockdown of CCT in A549 cells and its effect on virus growth. (A) Western blot analysis of CCTβ in A549 cells treated with no (−) siRNA or treated with GFP- or CCTβ-specific siRNA (siGFP or siCCTβ, respectively). RanBP5 detected with a polyclonal RanBP5-specific antibody (Santa Cruz) was used as a loading control. Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the left. (B) Quantitation of Western blot analyses of the knockdown of CCTβ from panel A. CCTβ intensities in siRNA-treated cells were expressed as a percentage of intensities observed in cells not treated with siRNA, which was set to 100%. CCTβ levels were normalized to the levels of RanBP5. Bars represent standard deviations based on three independent experiments. *, P < 0.05, based on a one-sample Student's t test. (C) Growth curves of influenza A/WSN/33 virus in CCT-silenced A549 cells. Control cells not treated with siRNA (−) or cells treated with GFP- or CCTβ-specific siRNA were infected at an MOI of 0.001. At the indicated points postinfection (24, 36, and 48 h), samples were collected, and virus titers were determined by plaque assay in MDBK cells. The results shown represent an average of two independent experiments, with the range indicated. (D) Viability assay of CCT-silenced A549 cells. Cells treated as in panel A were stained with 1% trypan blue, and viable and dead cells were counted. Cell viability was expressed as the percentage of viable cells in the culture. Results shown are an average of two independent experiments, with the range indicated.