Figure 5.

Autophagy inhibition by Atg12 knockdown has no effect on cell survival in 67NR cells treated with cisplatin, LY294002 or rapamycin. 67NR cells expressing an inducible Atg12 shRNA or a nonsilencing shRNA were treated with doxycycline and then with 1 µM cisplatin, 30 µM LY294002 or 0.2 µM rapamycin (A) or at the indicated concentrations (B and C). In (A), protein was collected for western blot after an 8 h treatment ± CQ. In (B), viability was measured with MTS reagent (short-term assay) after 24 h (cisplatin) or 48 h (LY294002 or rapamycin). In (C), cells grown in the presence of doxycycline were treated for 24 h, the treatment was washed, replaced with fresh medium ± doxycycline (doxy) and cells were allowed to recover for 4 d (long-term, clonogenic assays). Graphs are normalized to 100% per treatment and show mean ± standard error from three independent experiments performed in triplicate.