Par-4 knockdown results in G1 arrest and increased p21 expression. (A) Mec-1 cell clones expressing control or Par-4–specific shRNA were stained with propidium iodide. Cell-cycle analysis was performed by flow cytometry. Histograms represent mean ± SE of 4 control shRNA clones and 5 Par-4 shRNA clones (G1, P ≤ .0001; S, P ≤ .0002; G2, P = .15 comparing control to Par-4 shRNA-expressing clones). (B) shRNA lentivirus-infected cells were collected and total protein was isolated. Par-4 and p21 proteins were measured through immunoblot analysis. Protein quantifications were normalized to β-actin. (C-D) RNA was isolated from shRNA lentivirus-infected cells. Par-4 mRNA (C) and p21 mRNA (D) were quantified by qRT-PCR and were normalized to human 18S expression (C, P ≤ .0001; D, P ≤ .003 comparing control shRNA clones to Par-4 shRNA clones). (E) Human CLL cells were electroporated with CD79a siRNA and incubated for 72 hours. CD79a, Par-4, and p21 mRNA levels were quantified by qRT-PCR and were normalized to human 18S. Results represent mean ± SEM of triplicate determinations and an average of 4 CLL patient donors.