(A) Relative Pten mRNA levels of the indicated tissues. Bars represent mean ± SD (n = 3 per genotype). Statistical significance was determined by the two-tailed Student's t test.

(B) Immunoblot of the indicated proteins in growing cultures of primary MEFs. Each lane corresponds to an independent MEF preparation.

(C) Lysates from normal growing (C), serum starved (−), or starved and stimulated with insulin (+) immunoblotted to detect the indicated proteins.

(D) Primary MEFs (2 × 104) were plated at very low density, and the number of colonies was scored 3 weeks after. Bars represent mean ± SD (n = 3 independent MEF preparations per genotype). Representative plates are shown in the top. Statistical significance was determined by the two-tailed Student's t test.

(E) Primary MEFs were serially cultured (according to the 3T3 protocol) and the number of accumulated population doublings is scored at each passage. Values correspond to the mean ± SD (n = 3 independent MEF preparations per genotype). Statistical significance was determined by the two-tailed Student's t test.

(F) Neoplastic foci in MEFs retrovirally transduced with the viral oncoprotein E6 and either one of the indicated PI3K-dependent oncogenes, EGFRL858R or polyoma virus middle-T. Bars represent mean ± SD (n = 3 independent MEF preparations per genotype). Representative plates are shown in the top. Statistical significance was determined by the two-tailed Student's t test.

(G) Chemical carcinogenesis with 3-methyl-cholanthrene (3MC). Tumor-free Kaplan-Meier curves. Time is post-3MC injection. Statistical significance was determined by the log rank test.

(H) Incidence of cancers. Percentage of mice with one malignant tumor, or more (2–4), as indicated (left). Percentage of mice with the indicated type of tumor (right). Statistical significance was determined by the Fisher's exact test.