Figure 4. Akt3 regulated anchorage-independent growth and invasiveness of PMAs and GBM cells.

(A) Pten^cKO;p53^cKO;EGFRvIII PMAs were transduced with control (pLKO) or Akt3 shRNA-expressing lentivirus along with control (YFP) or retrovirus over-expressing Akt3 or kinase-dead Akt3 transcripts resistant to the shRNA (Akt3 rescue and K177A) or Akt1. Anchorage-independent growth was assessed by plating in soft agar. Left: Overlays of phase contrast and GFP fluorescence of representative colonies after 10 days. Scale bar = 50 μm. Right: Quantification, n= number of individual cultures per group. (B) Western analyses of cells in (A) using the indicated antibodies prior to plating in soft agar. (C) Quantification of colonies formed after human GBM cell lines T98G and U87-MG were transduced with control (pLKO) or AKT3-specific shRNA lentivirus and plated in agar from three independent experiments. (D) Akt3, but not Akt1 or Akt2, knockdown decreased invasion of Pten^cKO;p53^cKO;EGFRvIII PMAs. Quantification of cell invasion through matrigel in a Boyden chamber from three independently isolated cultures. Graphs in A, C and D indicate mean ± SEM and significance values (p) were calculated using unpaired t-tests.