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Fig. 3 | BMC Cancer

Fig. 3

From: A lowered 26S proteasome activity correlates with mantle lymphoma cell lines resistance to genotoxic stress

Fig. 3

Cyclin D1 accumulates after genotoxic stress in REC1 cells. a. JeKo1, REC1, NCEB1 cells were treated with 4 μg/ml etoposide for 24 h or vehicle, as a control, and harvested. Western blots were performed as described in the legend of Fig. 2 using Abs anti-cyclin D1, −cyclin D2 and -β-actin Abs. The anti-cyclin D2 Ab detects also cyclin D1, the specific cyclin D2 band is arrowed on the figure. The level of cyclin D1 in vehicle and etoposide conditions was estimated by densitometry and reported on the graph. b. Cells were cultured and treated as before and analyzed by western blot with the indicated Abs. Anti-GAPDH Ab served as a control for gel loading and transfer. c. REC1 cells were treated with 4 μg/ml etoposide for 24 h or vehicle, as a control, and harvested. We purified proteins either from whole cell extracts (w), or from cytoplasm (c), membranes (m) and nucleus (n) compartments. After SDS-PAGE separation and membrane transfer, blots were incubated with an anti-cyclin D1 Ab. The purity of each fraction was verified with Abs specific for cytosolic protein (HSP90), membrane protein (BCL2) and nuclear protein (PARP). d. NCEB1, JeKo1 and REC1 cells were treated with vehicle (0) or 4 μg/ml etoposide for 24–72 h. The cells were stained with PI and analyzed by FACS (Gallios, Beckman Coulter). Data were processed with the Kaluza software (Beckman Coulter). The percentage of cells within each phase of the cell cycle (sub-G1, G0/G1, S and G2/M) is indicated on the histogram. At least, 104 events were gated for each cell for each culture condition. The experiment has been carried out twice. e. MCL cells were treated with vehicle (−) or 4 μg/ml etoposide for 24 h (+) and harvested. Western blots were done as before with the indicated Abs

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