Fig 6. Loss of Nrf1 influences recovery of proteasome activity and cancer cell apoptosis after proteasome inhibition.

(A) HT29 cells were treated with either MG132 (2 μM), YU101 (100 nM) or Bortezomib (40 nM) for 1 hr after which the drugs were washed off and the cells were allowed to recover in the absence or presence of 50 μg/ml cycloheximide (CHX). Cells were frozen at different time points as indicated and were used for proteasome activity assays as described in Materials and Methods. For each time point, the results are normalized to DMSO-treated control in the case of hatched lines and to CHX-treated control in the case of solid lines. Error bars denote SD (n=3). (B) WT and Nrf1^-/- MEFs were treated with either MG132 (50 μM) or YU101 (300 nM) for an hour after which the drugs were washed off and proteasome activity was measured for cells collected at different time points as indicated. The results were normalized to DMSO-treated control for each cell type. Error bars denote S.E.M. (n=2). (C) MDA-MB-231 and U2OS cells were transduced with retrovirus expressing either sh-Nrf1 or vector control, and 48 hrs later were seeded in to 96-well plates. The next day, the cells were treated with indicated concentrations of YU101 and the pan-caspase inhibitor Z-VAD-FMK, and further incubated for 72 hrs. The cell viability was then assessed using the CellTiter-Glo method. The results were normalized to DMSO-treated control which was set to 100% in each case. Error bars denote S.E.M. (n=2). (D) MDAMB-231 and U2OS cells were transduced with retrovirus expressing either sh-Nrf1 or vector control, and 72 hrs later were treated with indicated concentrations of YU101 for 48 hrs (MDA-MB-231) or 24 hrs (U2OS). The cell lysates were used for immunoblotting to detect the levels of Nrf1 (with the antibody raised against N-terminus) and cleaved caspase-3. β-actin protein levels were used as loading control.