![Figure 2. Inhibition of Notch by GSIs stimulates apoptosis and blocks cell proliferation in CTCL cell lines. (A) SeAx cells were treated with GSI I (0.05-5μM) or the vehicle (DMSO) and analyzed with Western blot for activated Notch1 to Notch4 and Hes, as in Figure 1. (B-C) Induction of proapoptotic caspases 3 and 7 after treatment of MyLa by different GSIs: I, IX, XX, and XXI. The cells were treated by the increasing concentrations of GSIs for 48 hours (B) or with 5μM GSI I and 20μM of GSI IX, XX, and XXI for 0 to 72 hours (C). Data are fold increase over the residual caspase 3/7 activity in the control, vehicle-treated cells. P < .05 in both graphs for GSI I treatment compared with vehicle treatment. P < .05 for all tested GSI XX concentrations and for GSI XXI 20μM. P < .05 for GSI IX and XXI after 24 hours, for GSI XXI after 48 hours, and GSI XXI after 72 hours in MyLa. Data are mean ± SD. (D) Sub-G1 population in MyLa, SeAx, and Hut78 cells, which were treated with 5μM GSI I (0, 3, 24, and 48 hours). DNA was stained with NiM-DAPi, and the cells were analyzed by flow cytometry. Bars represent mean values. The experiment was repeated 3 times with similar results. *P < .05. (E) MyLa was treated with GSI I (5μM; 0, 3, and 24 hours) and stained with annexin V and PI for flow cytometry, as described in “Cell viability, proliferation and apoptosis.” Cells were gated according to the annexin V (green FL1 channel, x-axis) and PI-specific (red, FL3 channel, y-axis) fluorescence. Values in the quadrants represent the percentages of cells. The experiment was repeated twice with similar results. (F) PARP cleavage in MyLa, SeAx, and Hut78 cells treated with 5μM GSI I for 24 hours. Whole-cell extracts were prepared for Western blot as in Figure 1, and the blots were probed with the antibody against PARP detecting its intact form (116 kDa) and the caspase-cleaved 89-kDa fragment. (G) Cell proliferation of MyLa, SeAx, and Hut78 cells treated with increasing concentrations of GSI I for 24 hours. Sixteen hours before harvest, 3H-thymidine (1 μCi [0.037 MBq]/well) was added, and the results are expressed as mean counts per minute of triplicate experiments ± SD. *P < .05.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/14/10.1182_blood-2009-12-260216/4/zh89991057210002.jpeg?Expires=1724260582&Signature=xfftCiXmgU6W72xw3snpYNNXIpzaKFp0akcwOGfc1bFHlkRRJqyLUr56UEptQHX32~JCInv~Hx90S-mvAT9uHUYif-9n6WMcfs9da~q0TcxBqGzyfMuUPHz2v3~LMwE2d7Ad~o5EpDocX2bdNxCh44gqOtraoOXuVgswS9KQQe5zWfIxWxSqOeUkMXsAOKi-F7u8Fkr2V1jVzRdtLz6IQo9QA-K4Sl1EijNPzY8e5RpX~4ufYSjNYw0ZveTyDMwKzQwKuMvnw4VxoKvsGuaKAQDEkIDVuJ2czpgcxcJagqPvY00Mgg1Ypev-8v~UfZQFYmmPEY-j56yFba3NrNuTWA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inhibition of Notch by GSIs stimulates apoptosis and blocks cell proliferation in CTCL cell lines. (A) SeAx cells were treated with GSI I (0.05-5μM) or the vehicle (DMSO) and analyzed with Western blot for activated Notch1 to Notch4 and Hes, as in Figure 1. (B-C) Induction of proapoptotic caspases 3 and 7 after treatment of MyLa by different GSIs: I, IX, XX, and XXI. The cells were treated by the increasing concentrations of GSIs for 48 hours (B) or with 5μM GSI I and 20μM of GSI IX, XX, and XXI for 0 to 72 hours (C). Data are fold increase over the residual caspase 3/7 activity in the control, vehicle-treated cells. P < .05 in both graphs for GSI I treatment compared with vehicle treatment. P < .05 for all tested GSI XX concentrations and for GSI XXI 20μM. P < .05 for GSI IX and XXI after 24 hours, for GSI XXI after 48 hours, and GSI XXI after 72 hours in MyLa. Data are mean ± SD. (D) Sub-G1 population in MyLa, SeAx, and Hut78 cells, which were treated with 5μM GSI I (0, 3, 24, and 48 hours). DNA was stained with NiM-DAPi, and the cells were analyzed by flow cytometry. Bars represent mean values. The experiment was repeated 3 times with similar results. *P < .05. (E) MyLa was treated with GSI I (5μM; 0, 3, and 24 hours) and stained with annexin V and PI for flow cytometry, as described in “Cell viability, proliferation and apoptosis.” Cells were gated according to the annexin V (green FL1 channel, x-axis) and PI-specific (red, FL3 channel, y-axis) fluorescence. Values in the quadrants represent the percentages of cells. The experiment was repeated twice with similar results. (F) PARP cleavage in MyLa, SeAx, and Hut78 cells treated with 5μM GSI I for 24 hours. Whole-cell extracts were prepared for Western blot as in Figure 1, and the blots were probed with the antibody against PARP detecting its intact form (116 kDa) and the caspase-cleaved 89-kDa fragment. (G) Cell proliferation of MyLa, SeAx, and Hut78 cells treated with increasing concentrations of GSI I for 24 hours. Sixteen hours before harvest, 3H-thymidine (1 μCi [0.037 MBq]/well) was added, and the results are expressed as mean counts per minute of triplicate experiments ± SD. *P < .05.
