PKC inhibitors enhance anti-IgM induced apoptosis in CLL cells. (A-B) CLL cells were cocultured with bone marrow stromal cells in the presence and absence of 4μM ruboxistaurin (Rub.) or 4μM sotrastaurin (Sotr.). After 24 hours, an aliquot was taken for Western blot analysis of PTPN22 expression, and the remaining cells were split and cultured for an additional 48 hours with or without 10 μg/mL goat F(ab′)₂ anti–human IgM (sol-aIgM). Percentage of viable and dead cells was determined by annexin V/PI staining. (A) One representative experiment. (B) A summary of the results from 18 different experiments. Wilcoxon signed-rank test was used to evaluate significance of differences in leukemic cell viability. (C) The relationship between PTPN22 expression and the capacity of PKC inhibitors (PKCi) to enhance the cytotoxic effect of sol-aIgM was evaluated through Pearson rank correlation. The capacity of PKCi to enhance the cytotoxic effect of sol-aIgM (% PKCi + sol-aIgM cytotoxicity) was defined as the increase in the percentage of apoptosis induced by sol-aIgM + PKCi relative to sol-aIgM alone. The values were normalized against the percentage of viable cells at 48 hours to account for spontaneous apoptosis. The following formula was used: % PKCi + sol-aIgM cytotoxicity = [(%Viable_PKCi − %Viable_{sol-aIgM+PKCi}) / %Viable_PKCi] × 100 − [(%Viable_unst − %Viable_sol-aIgM) / %Viable_unst] × 100. The exact values are provided in supplemental Table 2. The percentage PKCi + sol-aIgM cytotoxicity was plotted against the amount of PTPN22 at 24 hours in cells without PKCi.