Figure 2.

Unc-51-like kinase 1 (ULK1) upregulation after DNA damage is dependent on p53. (a) Three U2OS clones containing tetracycline (Tet)-responsive p53shRNA (Tet-p53shRNA-clone1 and clone2) or a nonspecific shRNA (Tet-LashRNA) were treated with (lanes 3, 4, 7, 8, 11 and 12) or without (lanes 1, 2, 5, 6, 9 and 10) 1 μg/ml Tet for 2 days to knock down p53 or La, and then treated with (lanes 2, 4, 6, 8, 10 and 12) or without (lanes 1, 3, 5, 7, 9 and 11) 0.5 μM CPT for 2 days. The cells were then collected and lysed to do western blot. The blots were probed with antibodies against ULK1, p53 and p21. Actin was used as a loading control. (b) The same cells were treated as in (a) before doing the long-lived protein degradation (LLPD) assay. (c) Two U2OS clones containing Tet-responsive p53 (Tet-p53-clone1 and -clone2) were treated with (lanes 2 and 4) or without (lanes 1 and 3) 1 μg/ml Tet for 1 day to induce the expression of p53. The cells were then lysed to do western blot to test the expression level of ULK1, p53 and p21. Actin was used as a loading control. (d) One of the clone (clone1) as in (c) was transfected with either Luciferase small interfering (si)RNA (Luc, lanes 1–4) or ATG13 siRNA (ATG13i1, lanes 5 and 6) as described in Materials and Methods. After 3 days of siRNA transfection, p53 expression was induced with 1 μg/ml of Tet (lanes 2, 4 and 6) for 1 day. 3-MA (5 mM) was used to pretreat the cells for 1 h (lanes 3 and 4) before doing the LLPD assay. (e) The same clone (clone 1) as in (d) was either induced to express p53 (lane 2) or treated with 0.5 μM CPT for 2 days (lane 3), after which total RNAs were extracted from these cells. Quantitative PCR was used to test the mRNA expression levels of ULK1. These data are presented as mean±standard deviation (S.D.) and are representative of at least two independent experiments