Par-4 knockdown in Mec-1 CLL cells results in reduced growth in vitro and in vivo. (A) Western blot showing a reduction in Par-4 in Mec-1 cells expressing Par-4–specific shRNA. Par-4 protein values were normalized to β-actin. (B) Par-4 mRNA is decreased in Mec-1 cells expressing Par-4 shRNA compared with those expressing a control shRNA. Par-4 mRNA expression was normalized to human 18S and was measured in triplicate. (C) Growth curve of Mec-1 cells expressing control or Par-4–specific shRNA. Curves represent an average of 4 control and 4 Par-4 shRNA-treated clones. Slopes of the curves are different (P = .0024) as calculated by linear regression analysis. (D) Mec-1 cells (2 × 10⁶) treated with control or Par-4–specific shRNA lentivirus were engrafted into NSGS mice (n = 6) subcutaneously with Matrigel. Images represent 2 mice of 6 after 35 days of growth (left). Tumor volumes are plotted as a function of time (middle panel). Tumor volumes were calculated by measuring length and width with a caliper. Difference between the slopes of the 2 lines was found to be statistically significant (P ≤ .001) by linear regression analysis. After 35 days of growth, the tumors were excised and western blot analysis was performed to confirm tumors retained Par-4 knockdown in each mouse throughout the experiment (right panel). The numbers above blots refer to individual mice. Error bars represent SEM. L, left; R, right.