Figure 2
Endogenous p63 is required for survival in breast cancer cells.
(A) Knockdown of endogenous p63–induced Puma, Noxa, and PARP-1 cleavage, as assessed by immunoblot of the indicated cells 72 hours after infection with lentiviral shRNA vectors targeting 2 distinct p63 sequences (p63si-1 and p63si-2) or a nonspecific sequence (si-NS). None of these effects were observed in MCF-7 cells, which do not express abundant p63. (B) Apoptotic morphology following lentiviral p63 knockdown in HCC-1937 cells. Photomicrographs were taken 72 hours after lentiviral shRNA infection. Original magnification, ×100. (C) Apoptosis was observed following endogenous p63 knockdown, as assessed by annexin V/PI staining of unfixed cells 72 hours following infection with the indicated lentiviral shRNA vectors. Percentages indicate apoptotic cells (annexin V–positive and/or PI-positive). (D) Apoptosis following p63 inhibition was specific to breast cancer cells expressing abundant p63. Shown are results of annexin V/PI staining for 3 independent experiments. Error bars represent SD. (E) Puma and Noxa were induced following p63 knockdown. RNA was prepared 72 hours following lentiviral infection and assayed by QRT-PCR for the indicated genes. Error bars represent SD for 2 independent experiments, each performed in duplicate.
