Figure 6.

Synoviolin directly regulates p53-dependent apoptotic pathway in Drosophila fly. (A) Fly homolog of Synoviolin ubiquitinates fly homolog of p53 in vitro. GST-fusion Drosophila melanogaster p53 (Dmp53) was incubated with or without ATP, His-PK-HA-Ub, His-E1 (human), His-UbcH5C (human) and Drosophila Synoviolin (dSyno)ΔTM. Ubiquitinated proteins were probed with anti-HA antibody. (B) P53 protein level of wing discs was determined by immunostaining using anti-Dmp53 antibody (left). 2 representative pictures of each fly are shown. The fluorescence intensity of each 15 fly disc was quantified, and the net density level (Density INT/mm²) was determined by subtracting the density level of the background area (anterior half of disc) from the measured level of the target area (posterior half of disc) (right). Data are mean±s.e.m. of n=15. (C) Apoptosis was examined by Acridine orange staining of wing disc. Overexpression of dSyno in the posterior half of the discs reduced Dmp53 overexpression-induced apoptosis. (D) Overexpression of dSyno suppressed the Dmp53-induced bubbled wing phenotype. The extent of wing bubble (^*) in e16E-Gal4/UAS-Dmp53 flies varied with age, but the penetrance of bubbled wing phenotype was close to 100% (Supplementary Table 1). Overexpression of dSyno suppressed the Dmp53-induced bubbled wing phenotype, but dSyno-induced wrinkled phenotype at the posterior edge of wing was still observed (arrow).