Figure 6.

Lck inhibition enhances glucocorticoid sensitivity and apoptosis. (a) WEHI7.2 cells were treated with vehicles (0.1% ethanol and 0.01% DMSO), 10⁻⁶ M dexamethasone, 100 nM dasatinib, or both for 24 h. Cells were transferred onto a 96-well plate, treated with MTS reagent, and incubated for 3 h. Values represent the percent of control (Abs at 490 nm) and were obtained in triplicate. Inset, IC₅₀ for dexamethasone treatment alone or dexamethasone and dasatinib. (b) Cells were treated as in a and apoptosis was measured by flow cytometric analysis of Annexin V-positive cells. (c) Cells were treated as in a, lysed in non-denaturing buffer, and Caspase-3 activity was assessed by adding Ac-DEVD-AMC fluorogenic substrate. Values represent fold increase in fluorescence and were obtained in triplicate. (d) Cells were treated as in a, stained with Hoechst 33342, and visualized by epifluorescence microscopy (Zeiss axiovert S100) using a Zeiss × 40 fluorescent oil objective. Representative fields are shown for each treatment. Apoptotic nuclear morphology is indicated by white arrows. (e) WEHI7.2 cells were transduced with lentiviral shRNAs to selectively target Lck. Top: western blot for Lck and Fyn in cells transduced with empty vector or Lck shRNA. Bottom: control or Lck knockdown cells (2.0 × 10⁵ cells per ml) were treated with vehicle (0.1% ethanol) or dexamethasone for 24 h. Apoptosis was measured as in b. Data are representative of multiple independent experiments. Error bars represent the S.E.M.