Table 2. Effect of Notch1 and Notch2 silencing on CLL cell viability and Mcl-1 protein expression.
| Patients | siRNA | Receptor | % reduction in Notch expression | % reduction in CLL cell viability | % reduction in Mcl-1 protein expression | |
|---|---|---|---|---|---|---|
| TM | IC | |||||
| CLL 1 | ||||||
| siNotch1 | Notch1 | 55.0 | 67.0 | 32.6 | 39.0 | |
| siNotch2 | Notch2 | 51.2 | 61.1 | 17.9 | 38.0 | |
| CLL 2 | ||||||
| siNotch1 | Notch1 | 76.0 | 53.8 | 19.8 | none | |
| siNotch2 | Notch2 | 30.2 | 38.1 | 16.2 | none | |
| CLL 3 | ||||||
| siNotch1 | Notch1 | 98.0 | 99.3 | 65.2 | 70.0 | |
| siNotch2 | Notch2 | 71.0 | 64.0 | 51.4 | 29.8 | |
| CLL 4 | ||||||
| siNotch1 | Notch1 | 87.0 | 69.5 | none | none | |
| siNotch2 | Notch2 | 84.0 | 64.4 | none | none | |
| CLL 5 | ||||||
| siNotch1 | Notch1 | 57.0 | 80.1 | 32.7 | 36.9 | |
| siNotch2 | Notch2 | 43.7 | 65.3 | 35.1 | 51.0 | |
| CLL 6 | ||||||
| siNotch1 | Notch1 | 61.5 | 52.8 | 38.1 | 44.4 | |
| siNotch2 | Notch2 | 84 | 78 | 43.7 | 50.9 | |
| CLL 7 | ||||||
| siNotch1 | Notch1 | 73.2 | 89.1 | 29.7 | 44.2 | |
| siNotch2 | Notch2 | 69.0 | 56.7 | 23.1 | 32.6 | |
| CLL 8 | ||||||
| siNotch1 | Notch1 | 78.2 | 72.1 | 29.3 | 36.0 | |
| siNotch2 | Notch2 | 52.0 | 47.3 | 24.8 | 23.2 | |
| CLL 9 | ||||||
| siNotch1 | Notch1 | 67.0 | 73.0 | 33.8 | 27.7 | |
| siNotch2 | Notch2 | 81.0 | 72.5 | 39.5 | 34.0 | |
| CLL 10 | ||||||
| siNotch1 | Notch1 | 78.0 | 58.0 | 25.0 | none | |
| siNotch2 | Notch2 | 75.0 | 51.0 | 28.4 | none | |
| CLL 11 | ||||||
| siNotch1 | Notch1 | 48.0 | 59.0 | 20.8 | 22.9 | |
| siNotch2 | Notch2 | 41.0 | 54.0 | 38.2 | 41.7 | |
| CLL 12 | ||||||
| siNotch1 | Notch1 | 51.0 | 65.0 | 31.5 | none | |
| siNotch2 | Notch2 | 35.3 | 40.1 | 17.1 | none | |
| CLL 13 | ||||||
| siNotch1 | Notch1 | 31.0 | 35.2 | 17.7 | 23.0 | |
| siNotch2 | Notch2 | 75.7 | 61.2 | 16.1 | 48.0 | |
| CLL 14 | ||||||
| siNotch1 | Notch1 | 79.0 | 72.0 | 26.1 | 92.0 | |
| siNotch2 | Notch2 | 88.0 | 98.3 | 20.7 | 80.0 | |
| CLL 15 | ||||||
| siNotch1 | Notch1 | 44.4 | 52.0 | 58.4 | 41.9 | |
| siNotch2 | Notch2 | 72.7 | 59.8 | 34.9 | 26.0 | |
| CLL 16 | ||||||
| siNotch1 | Notch1 | 58.0 | 69.0 | 43.9 | 59.4 | |
| siNotch2 | Notch2 | 65.0 | 71.0 | 36.1 | 50.0 | |
| CLL 17 | ||||||
| siNotch1 | Notch1 | 47.1 | 53.0 | none | none | |
| siNotch2 | Notch2 | 69.0 | 71.0 | none | none | |
| CLL 18 | ||||||
| siNotch1 | Notch1 | 89.0 | 74.5 | 23.9 | 52.9 | |
| siNotch2 | Notch2 | 73.9 | 52.8 | 31.3 | 71.6 | |
| CLL 19 | ||||||
| siNotch1 | Notch1 | 36.0 | 31.3 | 18.4 | 32.0 | |
| siNotch2 | Notch2 | 56.4 | 45.1 | 20.5 | 35.4 | |
| CLL 20 | ||||||
| siNotch1 | Notch1 | 95.3 | 71.0 | none | none | |
| siNotch2 | Notch2 | 50.7 | 45.4 | none | none | |
| CLL 21 | ||||||
| siNotch1 | Notch1 | 68.3 | 75.3 | 33.6 | 21.0 | |
| siNotch2 | Notch2 | 57.3 | 61.3 | 44.3 | 44.0 | |
| CLL 22 | ||||||
| siNotch1 | Notch1 | 45.6 | 61.0 | none | none | |
| siNotch2 | Notch2 | 81.9 | 82.3 | none | none | |
Expression of Notch1 and Notch2 and Mcl-1 was analyzed by western blot in CLL cells transfected with Notch1 (siNotch1), Notch2 (siNotch2) or control siRNA (siCtrl). The band intensities of Notch1 and Notch2 (TM and IC) and Mcl-1 were quantified by densitometric analysis and normalized to GAPDH. Values represent the percentage reduction of Notch1 and Mcl-1 expression in siNotch1 cells, and of Notch2 and Mcl-1 expression in siNotch2 cells, compared with siCtrl cells. Viable CLL cells were quantified by flow cytometry using Annexin V/PI staining, and the percentage reduction in viability of siNotch1 and siNotch2 cells was calculated relative to siCtrl. None indicates reductions in cell viability or Mcl-1 expression lower than 5%.