Expression of APRIL mRNA and protein. (A) Quantitative real-time RT-PCR was performed on RNA samples isolated from the blood mononuclear cells of patients with CLL before (left) and after (right) depletion of CD2⁺ and CD14⁺ cells. The lines connect the preisolation and postisolation levels of APRIL mRNA in each sample. The amount of APRIL mRNA detected is indicated in arbitrary units. The amount of APRIL mRNA detected in an equivalent number of U937 cells is 30 units (data not shown). (B) Quantitative real-time RT-PCR measurement of the average amount of APRIL mRNA detected in CD14⁺ cells (n = 4), NLCs (n = 11), purified CLL B cells (n = 11), or isolated CD19⁺ blood B cells of healthy donors (n = 3), as indicated at the bottom of the histogram, ± SD (^**the mean level of APRIL mRNA detected in NLCs was significantly greater than that found in isolated CLL B cells, P < .01). (C) Representative immunoblot data showing the expression of APRIL by NLCs, CD14⁺ blood mononuclear cells, CLL B cells, or isolated CD19⁺ blood B cells of healthy donors. Whole cell lysates were prepared as described in “Materials and methods.” The protein content was normalized to 20 μg and subjected to immunoblot analysis with antibodies specific for APRIL or β-actin using ECL-based detection. (D) An immunofluorescence picture of NLCs and CLL cells stained with phycoerythrin-labeled anti-CD19 mAb (red) and goat IgG anti-APRIL polyclonal antibody that was detected using a fluorescein-labeled anti–goat IgG (green). The nuclei are labeled blue with Hoechst 33342.