Figure 4.

CHX and z-DEVD-fmk regulate oligomerization of K-Ras, p27, BAD, Bax, and Bak, and N-Myc expression but not oligomerization is required for transformation. (a and b) TNF-α+CHX induces robust apoptosis as analyzed by DNA fragmentation (a), but TNF-α+CHX-generated blebbishields do not form spheres (b). P≥0.05 in panel (a) indicates no significant differences in DNA fragmentation between three CHX dose combinations (n=3) (comparison of black bars only). Inset in panel (b) shows early induction of blebbishields by TNF-α+CHX. No treatments were carried out during transformation phase. (c) CHX inhibits transformation of FasL+AZ58-generated blebbishields. No treatments were carried out during sphere formation phase. (d–f) CHX not only enhances mitochondrial oligomerization of K-Ras, p27, BAD, Bax, Bak, and pBAD¹¹² but also enhances Bax cleavage and release of Smac and cytochrome-C (d), resulting in differential caspase-9 cleavage (e) and loss of N-Myc and c-Myc (f). (g) N-myc is expressed in sphere-forming but not in non-sphere-forming (floating) blebbishields. *BS, blebbishields isolated after 24-h treatment; Spheres*, allowed transformation time 4 h. (h and i) Caspase inhibition abolishes transformation from FasL+AZ58-generated blebbishields (h) and fails to rescue sphere formation from TNF-α+AZ58- or TRAIL+AZ58-generated blebbishields (i). No treatments were carried out during sphere-formation phase. (j) Caspase inhibition blocks oligomerization of K-Ras.