Ectopic expression of CA-STAT5b leads to BCR multinucleated cells. (A) Surface BCR expression on CA-STAT5b–transduced (■) or control-transduced (●) PB B cells was determined in time by flow cytometric analysis of surface of kappa and lambda light chain expression. Cells were cultured on CD40L plus IL-2 and IL-4. Data represent means (± SD) of 3 independent experiments. Identical results were obtained with 12 different donors. Similar results were obtained with tonsil (n = 6). (B) Cytospins of CA-STAT5b⁺ tonsil B cells and L428 were prepared, and images in Figure 4B were visualized and captured on an Olympus BX51 light microscope with a 100×/1.3 oil objective. Giemsa staining was performed to visualize the nuclei of the cells at a total magnification of 1000×. The presence of mononucleated and multinucleated cells strongly resembled the Hodgkin and Reed-Sternberg cells, respectively (n = 6). (C) To enumerate the percentage of cells with multiple nuclei, DNA content was measured using propidium iodide staining and flow cytometric analysis. Single cells were gated and cells with more than 4N DNA were considered to be cells with multiple nuclei. Data represent means (± SD) of 3 CA-STAT5b⁺ tonsil B-cell lines in comparison with L428 and freshly isolated PB B cells.