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. 2006 Dec 14;26(1):113–122. doi: 10.1038/sj.emboj.7601490

Figure 5.

Figure 5

Synoviolin up-regulates p53 level in cells under normal and genotoxic stress conditions. (A) Synoviolin ubiquitinates p53 in vitro. (B) The ubiquitination of p53 is dependent on an intact RING finger domain. (C) WT Synoviolin, but not RING finger mutant, ubiquitinates p53 in vivo. (D) Synoviolin over-expression in HEK293 cells increases degradation of p53 (top). At 24 h after the transfection with empty vector, wild type Synoviolin (WT), mutants in RING finger domain (C307S) or p53 binding domain deletion mutant (Δ53BD), p53 expression was examined at indicated time of cycloheximide treatment. Degradation of p53 is inhibited in synoviolin knockout MEFs (Syno−/−) compared with wild type MEFs (Syno+/+). The p53 expression was examined after the indicated time of cycloheximide treatment. The remaining p53 expressions were normalized to β-actin expression and plotted against time (minutes). (E) Effects of knockdown of Synoviolin and/or other known E3 ubiquitin ligases for p53 on the level of p53 in RKO cells. Quantified p53 level is expressed as relative levels (1.0=GFP-siRNA treated RKO cells). (F) Genotoxic stress induces p53 accumulation in the absence of Synoviolin. GFP- siRNA (G). syno siRNA (S). At 48 h after transfection, the RKO cells were treated with vehicle, camptothecine (CPT, 0.5 μM), actinomycin D (ActD, 5 nM), X-Ray (9 Gray) or thapsigargin (TG, 1 μM) for 6 h. (G) Synoviolin knockdown sensitizes cells to genotoxic stress. At 48 h after transfection, cells were exposed to the indicated doses of ionizing-radiation for 24 h, followed by FACS analysis to detect annexin V-positive cells. Data in (D) and (G) are mean±s.e.m. of n⩾3. *P<0.05.