Figure 6

Knockdown of Akt1 and Akt3 but not Akt2 inhibits proliferation and tumor growth in K-RAS-mutated MDA-MB-231 cells. (a) Cells (3×10⁴) were plated in 6 cm culture dishes. At the indicated days after seeding, cells were counted and graphed. The data points represent the mean cell counts±S.E.M. of eight parallel experiments from two independent experiments. Asterisks indicate significant prolongation of PDT after knockdown of Akt1 and Akt3 compared with scramble-shRNA (scr-shRNA) (*P<0.05, ***P<0.001). (b) Protein samples were isolated from the cells counted on day 7 and expression of Akt isoforms was tested by immunoblotting. (c) Indicated cells (3×10⁴) were plated for 24 h and treated with DNA-PKcs inhibitor NU7441 (10 μM). Cells were count on day 6 after treatment and graphed. Data present mean cells numbers of eight data±S.E.M. obtained from two independent experiments. (d) Nude mice were injected with indicates cells (2×10⁶ cells) in both dorsal flank and tumor growth assay was performed as described in Materials and Methods section. Data present mean tumor volume±S.E.M. of 14 tumors (seven mice) inoculated with MDA-MB-231-expressing scr-shRNA and of 12 tumors from six animals inoculated with MDA-MB-231 cells expressing Akt1-, Akt2- or Akt3-shRNA. Asterisks indicate a significant tumor growth delay by knockdown of Akt1 as well as Akt3 (***P<0.001) and increased in tumor volume by knockdown of Akt2 (*P<0.05), measured 6 weeks after inoculation. (e) Representative images of tumors following inoculation of MDA-MB-231 cells expressing scr-shRNA as well as shRNA against the Akt isoforms.