Figure 2.

ABT-737 cooperates with Noxa to induce Bax/Bak-dependent killing

A: The viability of wild-type MEFs (WT), Bax/Bak-deficient MEFs (DKO), and Bak- or Bax-singly deficient MEFs was determined by PI exclusion 48 h after exposure to ABT-737 (10 μM) or Etoposide (10 μM).

B: ABT-737 is a Bad BH3 mimetic. Based on the relative affinities (IC₅₀ in nM) of ABT-737 for mammalian pro-survival proteins, determined in solution competition assays (Fig. S1A), ABT-737 and Bad bind to the same subset of Bcl-2 pro-survival proteins. According to our model for initiating the apoptotic program (Chen et al., 2005; Willis et al., 2005), Bad and Noxa are poor inducers of apoptosis individually because each binds only a subset of the pro-survival proteins, whereas Bim is a potent killer because it binds all of them. By this rationale, ABT-737 (like Bad) should also cooperate with Noxa to kill cells.

C: Noxa triggers Mcl-1 degradation. Immunoblots of lysates prepared from the MEFs after retroviral infection with wild-type Noxa or the 3E mutant (an inactive mutant that does not bind Mcl-1) probed for Mcl-1 and HSP70 (loading control).

D: Noxa sensitizes wild-type MEFs to ABT-737 killing. Wild-type MEFs expressing wild-type human Noxa or an inactive mutant (Noxa 3E) (Willis et al., 2005), were exposed to ABT-737 for 8 h and their viability determined.

E: Bax/Bak-deficient MEFs (DKO) are resistant to ABT-737 even when Mcl-1 is targeted. Long-term clonogenic survival of cells exposed to ABT-737. Equal numbers of the indicated MEFs, or their counterparts stably expressing Noxa or the inactive Noxa 3E, were plated in media containing vehicle or ABT-737 (1 μM, replenished after 3 d) and the colonies formed scored after 6 d. The number of colonies obtained with ABT-737 treatment is expressed as a proportion of colonies formed with the vehicle alone. - no colonies.

F: Either Bax or Bak can mediate killing by ABT-737 provided Mcl-1 is targeted. Viability of the indicated MEFs stably expressing Noxa was determined 8 h after exposure to ABT-737. Note that Bax/Bak-deficient MEFs (DKO) are resistant.

G: Noxa sensitizes FDC-P1 myeloid cells to ABT-737 killing. The viabilities of FDC-P1 cells, retrovirally infected to express Noxa, mutant Noxa 3E or Bad, were compared after a 24 h treatment with graded doses of ABT-737.

Data in A and D–G represent means ± SD from a representative of 3 experiments.