Inhibition and silencing of BCR signaling results in a decrease in Par-4 expression. (A) Mec-1 CLL cells were treated with control (Ctrl) or Lyn-specific shRNA lentivirus and selected through puromycin treatment. Cells were collected at day 17 and total protein and RNA was isolated. Left panel, Levels of Lyn protein. Right panel, Levels of Par-4 mRNA. (B) Human CLL cells were electroporated with CD79a siRNA and incubated for 24 hours. Total protein was isolated and probed for CD79a and Par-4. Western blot represents data from 1 CLL donor with average results for 4 other donors shown as histogram. Ctrl siRNA, n = 2; CD79a siRNA, n = 2. Error bars represent SEM. (C) Surface IgM levels were measured on human CLL cells electroporated with control and CD79a siRNA by flow cytometry (left, representative histogram for 1 patient; middle, average mean fluorescent intensity [MFI] of IgM expression for 4 donors). Total RNA was isolated from cells, and CD79a and Par-4 mRNA were measured by qRT-PCR. Expression values were normalized to human 18S expression performed in triplicate for 4 different donors. *P ≤ .05, **P ≤ .01, ***P ≤ .001 determined by the Student t test.