Figure 5. Disrupting αvβ3/Src signaling specifically drives PUMA expression in stem-like cells.

(A) PUMA immunoblot of LM2-4 cells treated with the indicated drugs for 24 hours. All drugs were dosed at 100 nM, except for bortezomib and docetaxel, which were added at a sublethal concentration of 5 nM. (B and C) Immunoblots of the indicated cell types after treatment with DMSO vehicle control (Veh) or 100 nM dasatinib (Das) for 24 hours. (D and E) Western blot analysis of LM2-4 cells stably expressing c-Src shRNA (shSrc) or a nonsilencing shRNA control (shCtrl) (D) or transiently transfected with mock, vector alone, full-length β3 cDNA (β3 FL), or a β3 Src–binding domain mutant (β3 759x) (E). (E) All lanes were run on the same gel, but the β3 759x lane was noncontiguous (black line). (F) qPCR analysis of ubiquitin ligases that regulate Slug expression. Fold change (2^–ΔΔCT) in BT474 cells stably expressing β3 cDNA is shown relative to vector control cells. β-Actin was used as a loading control. n = 3 independent experiments. Each sample was run in triplicate. Data represent the mean ± SEM. (G–J) Western blot analysis for the indicated proteins in BT474 cells stably expressing vector control, full-length β3 cDNA, or the β3 759x mutant (G); BT474 cells with transient siRNA knockdown of FBXO11 (H); LM2-4 β3–knockdown cells (I); and LM2-4 cells treated with 100 nM dasatinib (J). (A–E and G–J) β-Actin or Hsp90 was used as a loading control. Data shown are representative of 3 independent experiments. See also Supplemental Figure 5.