Figure 3

Cyclin D1 expression amplifies a caspase-dependent apoptosis. A. 8226- and LP1-derived clones were treated for 24 h with vehicle (Ctrl) or bortezomib (Bort, 5 or 10 nM). Cell viability was quantified in MTT assays. The absorbance (OD at 490 nm) of each clone treated with the drug is expressed relative to that of the corresponding clone treated with vehicle (defined as 1 in arbitrary unit, AU). For each set of culture conditions, the mean of triplicate ratios is indicated on the graph, together with the SD. ** p < 0.01. B. GFP- (in black) or D1-GFP-expressing clones (in gray) were treated with vehicle (Ctrl) or 10 nM bortezomib. After 24 h, the cells were stained with anti-APO2.7 Ab and analyzed by flow cytometry. At least 2 × 10⁴ events were gated for each clone and set of culture conditions. Representative profiles of APO2.7-stained cells are shown. The quantification of APO2.7-positive cells in several independent experiments is reported in the Additional file 3. C. Cells were treated with the pancaspase inhibitor the Q-VD-OPh (or vehicle) for 1 h before treatment with bortezomib (or vehicle) at the concentrations indicated for an additional 24 h. Cell viability was measured as described in A. Each experiment was carried out two or three times, in triplicate. ** p < 0.01. D. Cells were treated as described above for 24 h. Whole-cell protein extracts were obtained and separated by SDS-PAGE. Proteins were blotted and analyzed with the Abs indicated. The GAPDH Ab was used as a loading control. The proforms of caspase (Casp.) are indicated by gray arrows; the cleaved, activated forms of caspase are indicated by black arrows. Cleaved PARP is indicated by a dotted arrow. E. The cells were treated as described above for 24 h. Whole-cell protein extracts were prepared and separated by SDS-PAGE. Proteins were blotted and analyzed with the Abs indicated. The GAPDH Ab was used as a loading control. MCL1 protein is indicated by an arrow.