Akt1 in Mammary Development were asfollows: 59gatccccGCGACCAACGCCTTGATTGttcaa- gagaCAATCAAGGCGTTGGTCGCtttttgga-39 for luciferase, 59- gatccccGAGTTTGAGTACCTGAAGCttcaagagaGCTTCAGG- TACTCAAACTCtttttggaaa-39 for Akt1, and 59-gatccccgcgt Gat- agaggagagaatttcaagagaATTCTCTCCTCTATCACGCttttggaaa-39 for Btn1a1. Knockdown cells were maintained in 1.0 mg/ml puromycin. For cell proliferation 100,000 cells were plated in each well of a 6-well plate. Cell proliferation was determined by Trypan blue exclusion using a TC-10 automated cell counter (Bio-Rad, Hercules, CA), and calculating the percent change of in number of cells relative to cell number on day 0. Statistical analysis A Bartlett test for homogeneity followed by a two-tailed students t-test adjusted for equal variance were performed to test for statistical significance. For genotype and exposure interactions, a two-way ANOVA followed by a Tukey HSD test was performed. A P value of ,0.05 was considered to be statistically significant. Statistics were calculated using R software (R Project). Results Akt1 deficiency delays mammary gland postnatal development Previously identified roles for Akt1 in developmental delay and reproductive impairment [11,14,17,19,20] led us to examine its involvement in postnatal mammary gland development. We first examined the growth patterns of mammary glands in Akt1+/+ and Akt12/2 mice at different stages of postnatal development, including neonatal (PND 11), juvenile (PND 25), pubertal (PND 50), and adult (PND 70) stages (Fig. 1 A–H). Akt12/2 mammary glands exhibited significantly stunted ductal outgrowth during the juvenile and pubertal time periods (PND 25 and PND 50, respectively), but by adulthood (PND 70) the glands occupied the same amount of space in the fat pad compared to Akt1+/+ mice (Fig. 1 K). Accordingly, primary duct length was reduced in Akt12/2 glands at both PND 11 and PND 25 (Fig. 1 I). Analogous to the ductal outgrowth, at PND 50 growth past the lymph node was shorter in Akt12/2 glands (Fig. 1 J). At Figure 1. Akt1 promotes postnatal mammary gland growth. Representative whole mounts of Akt1+/+ (A–D) and Akt12/2 (E–H) mammary glands at PND 11 (A,E), PND 25 (B,F), PND 50 (C,G), and PND 70 (D,H). Measurement of primary duct length at PND 11 and PND 25 in Akt1+/+ and Akt12/2 glands (I). Measurement of the growth past the lymph node at PND 50 and PND 70 in Akt1+/+ and Akt12/2 glands (J). Quantification of ductal outgrowth for Akt1+/+ and Akt12/2 mammary glands at PND 11, 25, 50, and 70 (K). Data represented in line plot as mean +/2 standard error. Data represented in bar plots as mean + standard error. Scale bars indicate 1,000 mm. Statistical analyses were conducted using a two-tailed student’s t test. **p,0.01, *p,0.05. n = 3–8 per genotype, per age group. doi:10.1371/journal.pone.0024432.g001 PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24432