Figure 8: DBC1 sumoylation is required for p53-mediated apoptosis.

(a) shRNA-insensitive Flag-tagged DBC1 or its K591R mutant was expressed in HeLa cells that had been stably transfected with nonspecific shRNA (shNS) or shDBC1. After incubation with and without etoposide for 36 h, they were subjected to immunoblot with anti-DBC1, anti-acetyl p53, anti-cleaved PARP1 and anti-cleaved caspase-9 (Casp-9) antibodies. ‘c’ denotes cleaved (left). The band intensities of acetylated p53, c-PARP1 and c-Casp-9 from lysates were quantified, and those seen with both shNS and etoposide were expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n=3). (b) shNS or shDBC1 was expressed in HeLa cells with or without shSENP1. Cells were then incubated in the presence and absence of etoposide for 36 h (left). The band intensities of acetylated p53, c-PARP1 and c-Casp-9 were quantified as in a (right). Error bar, ±s.d. (n=3). (c) shSUMO2/3 or shPIAS3 was expressed in HeLa cells with Flag-SUMO3 or Myc-PIAS3 (left). The band intensities were quantified as in a (right). Error bar, ±s.d. (n=3). (d) HeLa cells expressing Flag-DBC1 or its K591R mutant were incubated with and without etoposide for 36 h. They were then subjected to TUNEL assay. Scale bars, 20 μm. (e) shNS or shDBC1 was expressed in HeLa cells with shNS, shSUMO2/3, shPIAS3 or shSENP1. After incubation with and without etoposide, cells were subjected to TUNEL assay. In a,c, ‘i’ and ‘e’ denote shRNA insensitive and endogenous, respectively. Scale bars, 20 μm.