Figure 2. Pten mutant primary cultures mirror the development of primary cultures expressing oncogenic Ras^V12.

(A–C) Wild type. (D–F) Ras^V12 (Act5C-Gal4; UAS-Ras^V12). (G–I) Pten¹¹⁷. (J–L) Pten¹¹⁷; Ras^V12 (Pten¹¹⁷; Act5C-Gal4/Pten¹¹⁷; UAS-Ras^V12). After five days, primary cultures of all genotypes (A, D, G, J) had differentiated cell types including fat body (open arrowheads) and muscle (arrowheads). After ten days, wild-type cultures (B) had only the same differentiated cell types, whereas, cultures of the other genotypes (E, H, K) had patches of spindle-shaped cells. After 20 days, wild-type cultures (C) had only the same differentiated cell types, whereas, cultures of the other genotypes (F, I, L) were densely populated with spindle-shaped cells. (M and N) Western-blot analysis of primary culture extracts with cells of the indicated genotypes. (M) The Akt pathway is activated (pAkt) above control (wild type) levels in cultures with Ras^V12 expressing cells (Ras^V12), Pten mutant cells (Pten¹¹⁷) and Pten mutant cells expressing Ras^V12 (Pten¹¹⁷; Ras^V12). The Erk pathway is activated (dpErk) above control (wild type) levels in cultures with Ras^V12 expressing cells (Ras^V12) and Pten mutant cells expressing Ras^V12 (Pten¹¹⁷; Ras^V12). Total Akt and Erk, as detected by α-Akt and α-Erk were used as loading controls [40], [41]. Akt is detected as two bands (Cell Signaling Technology) [31]. For unknown reasons the lower band is more prominent in the Pten mutant cultures.