Blk promotes the proliferation of malignant CTCL cells. (A,B) Malignant T cells (MF2000; 4 × 10³) were incubated with the given concentrations of (A) LckI or (B) PP1 for 48 hours, and the proportion of viable cells was determined by MTT assay as measured by the absorbance (Abs) at 560 nm. MTT determinations were performed with triplicate cultures, and the error bars represent SDs. (C) Malignant (MF2000) or nonmalignant (MF1850, MySi) T cells (4 × 10³) were cultured for 48 hours with the indicated concentrations of LckI. Then [³H]-thymidine was added, and the cells were cultured for 20 hours, before determination of [³H]-thymidine incorporation. The [³H]-thymidine incorporation was determined as mean counts per minute (CPM) of triplicate cultures. The percentage of proliferation of each cell line was calculated as the mean CPM for cells treated with a given concentration of LckI divided with the mean CPM for cells treated with vehicle. (D) MF2000 cells were incubated for 48 hours with vehicle (−), LckI (10 μM), Jak3I (40 μg/mL), or Ag1478 (200 ng/mL). Subsequently, the cells were stained with 7-AAD, and the percentage of 7-AAD–positive cells was determined by flow cytometry. (E) MF2000 cells were incubated with vehicle (−), LckI (10 μM), or Ag1478 (200 ng/mL) for 24 hours, and the levels of Cdk2 and Stat3 were examined by WB. (F) MF2000 cells were transiently transfected with nontargeting (NT) or Blk-specific siRNA and cultured with [³H]-thymidine. After 24 hours, the cells were harvested, and the [³H]-thymidine incorporation (left), as well as the levels of Blk and Erk expression (right), was determined. The [³H]-thymidine incorporation is expressed as the mean CPM of 6 replicate cultures, and the error bars represent SDs. (G) MF2000 cells were transiently transfected with NT or Blk-specific siRNA. At 24 hours after transfection, the cells were lysed, and the lysates were analyzed by WB (left). The band intensities from the WB were quantified, and the relative band intensities between MF2000 cells transfected with NT and Blk-specific siRNA are depicted (right). The data are representative of at least 3 independent experiments.