Figure 2

Effect of shRNA-induced bcl-xl , bcl -2, or mcl -1 silencing and of ectopic bax overexpression on A375 xenograft growth. siRNA-induced bcl-xl, bcl-2, or mcl-1 silencing was used for preliminary in vitro experiments, whereas cells stably expressing shRNAs were used for in vivo experiments (see under Methods). (A) western blots of Bax in A375 and A375/Tet-bax cells. (B, C, D) western blots of Bcl-xl, Bcl-2, and Mcl-1, respectively, in A375 cells transfected with bcl-xl siRNA, bcl-2 siRNA, mcl-1 siRNA, or non-specific (NS) siRNA. (E) in vivo growth of A375 (●), A375/bcl-xl-shRNA (■), A375/bcl-2-shRNA (○), and A375/Tet-bax cells (□). Western blots correspond to cultured transfected cells, and are similar to those obtained in extracts of xenografts (see Methods) 1 or 2 weeks after tumor inoculation (not shown), thus indicating stability of each plasmid expression. Tumor growth (E) was measured during a 3-week period. Data are means ± S.D. of 9-10 mice per group. The significant test refers, for all groups (E), to the comparison between A375/bcl-xl-shRNA, A375/bcl-2-shRNA, or A375/Tet-bax cells and A375 controls (* p < 0.01). A375/bcl-2-shRNA was also compared versus the A375/bcl-xl-shRNA group (⁺p < 0.01) (E). Rates of tumor growth obtained with A375/mcl-1-shRNA cells were not significantly different from those displayed for A375/Tet-bax cells (□) (E) (not shown). Rates of tumor growth obtained with A375 cells transfected with empty Tet-off vector or with NS shRNA were not significantly different from those displayed for untransfected A375 controls (●) (E) (not shown).