Figure 6

NSC114792 decreases cell viability and induces apoptosis in cancer cells with persistent JAK3 activation. (A) NSC114792 decreases cell viability only in L540 cells that express persistently-active JAK3. L540, HLDM-2, MDA-MB-468 and DU145 cells were treated with either vehicle (DMSO) alone, NSC114792 at different concentrations or the pan-JAK inhibitor AG490, and incubated for the indicated time periods. The trypan blue exclusion assay was performed to count viable cells. Results are shown as the mean of three independent experiments (± SD indicated by error bar). *, p < 0.001, indicates statistical significance compared to DMSO-treated cells. (B) TUNEL assay shows that NSC114792 induces apoptosis in L540 cells. Cells were treated with vehicle (DMSO) alone or NSC114792 at various concentrations for 72 hours. Cells were then stained with FITC-conjugated BrdU antibody and propidium iodide (PI), and subsequently subjected to flow cytometry. The percentages of TUNEL-positive cells are indicated. (C and D) Treatment with NSC114792 increases cleaved fragments of Poly (ADP-ribose) polymerase (PARP) and caspase-3, and decreases the expression of anti-apoptotic genes in a dose-dependent manner in L540 cells. Cells were treated with vehicle (DMSO) alone or NSC114792 at various concentrations for 48 hours. Whole-cell extracts were processed for Western blot analysis using antibodies specific for the molecules indicated. GAPDH serves as a loading control.