Effects of R788 treatment on BCR signaling, leukemic cell proliferation, and survival. (A) B6/C3H F1 mice with overt TCL1-002 leukemia were treated for 7 days with R788 or vehicle control. Peripheral blood samples were collected 30 minutes after the last treatment, and immunoblotting analysis was performed on cellular extracts using the indicated antibodies. Changes in the amount of phosphorylated Syk, BLNK, and ERK are summarized in the graph. The phospho-specific signal was calculated as fold difference in signal intensity relative to the signal in sample C1, which was arbitrarily set to 1.0. Values were normalized against total Syk, which was used as a loading control. (B) Ki67 staining was performed on peripheral blood samples from animals with overt TCL1-002 leukemia treated with vehicle control (n = 7) or R788 (n = 6), as described above. The percentage of Ki67-positive cells is indicated in each histogram plot. Pooled data and statistical evaluation are presented in the graph. Open circles represent the percentage of Ki67-positive cells in each animal; mean values and SD are indicated with filled circles and error bars, respectively. Statistical analysis was performed with the Mann-Whitney rank sum test. (C) In vivo BrdU labeling was done in animals with adoptively transferred TCL1-002 leukemia that were treated for 7 days with vehicle control or R788. BrdU was injected twice on day 7 together with R788 or vehicle control, and spleen samples were collected 6 hours later. Representative examples of frozen spleen sections stained with anti-BrdU antibody are shown. BrdU-positive cells are dark brown. The graph summarizes the data from the analysis of 4 mice treated with R788 and 4 mice treated with vehicle control. Four independent fields per spleen were counted using a Widefield Leica DMR microscope and NIH ImageJ software. Open circles represent the number of BrdU-positive cells/field; mean values and SD are indicated with filled circles and error bars. (D) Representative examples of paraffin-embedded spleen sections stained with TUNEL-peroxidase from mice treated for 7 days with R788 or vehicle control. The graph summarizes the data from the analysis of 4 mice treated with R788 and 3 mice treated with vehicle control. Open circles represent the number of TUNEL-positive cells/field; mean values and SD are indicated with filled circles and error bars.