and incubated for 60 minutes at room temperature in horseradish peroxidase conjugated goat anti-rabbit IgG (1:2000, Cell Signal- ing) or goat anti-mouse IgG (1:10,000 Millipore) diluted in 5% milk/TBST. Membranes were washed 3 times (5 minutes per wash) in TBST and once in TBS prior to visualization using enhanced chemiluminescence (Thermo Scientific). Determination of apoptotic rates by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and quantitation PND 70 mammary glands were stained for apoptosis using the ApopTag kit (Chemicon, Temecula, CA) according to the manu- facturer’s instructions. Tissues were counterstained with hematox- ylin. Digital images were captured using the Aperio Scanscope Imagine System (Aperio Technologies, Vista, CA), and TUNEL positive nuclei were measured using the nuclear algorithm analysis on the Aperio Imagescope v10.2. At least 200 cells per animal were measured. RNA isolation RNA was isolated from thoracic mammary glands from mice (PND70) previously flash frozen in liquid nitrogen or MCF7 cells using the mirVANA RNA Isolation Kit (Ambion Inc., Austin, TX) according to manufacturer’s protocols. RNA con- centration was quantified using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), aliquoted, and stored at 280uC until needed. RNA microarray hybridization Integrity of RNA isolated from thoracic mammary glands was assessed using an Agilent 2100 Bioanalyzer (Agilent Tech- nologies, Palo Alto, CA). Complementary DNA (cDNA) was synthesized from 300 ng of total RNA and purified according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA.). Equal amounts of purified cDNA were used as the template for subsequent in vitro transcription reactions for complementary RNA (cRNA) amplification and biotin labeling using the Affymetrix RNA Transcript Labeling Kit. cRNA was purified and fragmented according to the protocol provided with the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). All GeneChip microarrays (Mouse Genome 430 2.0 Array) were hybridized, washed, stained, and scanned using the Com- plete Gene Chip Instrument System according to the Affymetrix Technical Manual. The Mouse Genome 430 2.0 microarray contained 17 probe sets. Microarray data analysis All data is MIAME compliant and data is publically available at GEO with accession number: GSE30671. Affymetrix CEL files were imported into PartekH Genomics SuiteTM 6.5 (Partek Inc., St. Louis, MO) using the default Partek normalization parame- ters. The robust multiarray average (RMA) normalization method was used to normalize data. Statistically significant differentially expressed genes between Akt1+/+ and Akt12/2 mammary glands were defined as having a p,0.05 as determined by a one-way analysis of variance (ANOVA). The recovered P- values of the statistically significant genes were corrected using a step-up false discovery rate value of 5%. The final list of differentially expressed genes included only those genes with a 1.5-fold change (up or down) with a p,0.05. Data was further analyzed using Ingenuity Pathways Analysis (IPA) (IngenuityH Systems, www.ingenuity.com). The IPA Functional Analysis program was used to identify the biological functions and/or Akt1 in Mammary Development diseases that were most significant to the data set. Only molecules from the dataset meeting the p value cutoff of 0.05 and were associated with biological functions and/or diseases in Ingenuity’s Knowledge Base were considered for the analysis. Right-tailed Fisher’s exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set was due to chance alone. IPA’s Pathway Analysis generated graphical representation of the molecular relationships between molecules, represented as nodes, and the biological relationship between nodes, represented as an edge (line). All edges were supported by at least 1 reference from the literature or from canonical information stored in the Ingenuity Pathways Knowledge Base. Nodes are displayed using various shapes that represent the functional class of the gene product, while the intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Edges are displayed with labels describing the nature of the relationship between the nodes. Quantitative RT-PCR Total RNA (1 mg) was treated with DNase (Promega, Madison, WI) and reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s protocols. cDNA templates were amplified with pre-optimized mouse-specific QuantiTectH primer assays (Qiagen, Valencia, CA) using iQ SYBR Green Supermix (Bio-Rad) on an iCycler iQ Multicolor Real-time PCR Detection System (Bio-Rad). Each 25 ml sample was run in triplicate, and mRNA levels were analyzed relative to hypoxanthine phosphoribosyltransferase (HPRT). Log2-trans- formed relative expression ratios were calculated as described using the equation set forth by Pfaffl (Pfaffl 2001). Pseudopregnancy stimulation Female mice at 60–90 days old were injected intraperitoneally (i.p.) with 5.0 international units (IU) of PMSG (Sigma Aldrich) followed by 5.0 IU of hCG i.p. 48 hours later. Mammary glands were excised from mice 18 hours after hCG administration, thoracic glands were flash frozen for RNA, and inguinal glands were either processed for whole mounts or fixed in 10% neutral buffered formalin. Immunofluorescence Indirect immunofluorescence was performed on formalin-fixed, paraffin-embedded 7 mm sections. Sections were re-hydrated, and antigen retrieval was performed by heating slides in 10 mM sodium citrate in a steamer for 20 minutes. Sections were blocked overnight with normal goat and rabbit serum (Vector Laboratories, Burlingame, CA), and incubated with primary antibodies against Btn1a1 (AV45164, Sigma-Aldrich) and Akt1 (sc-1618, Santa Cruz) followed by Alexa Fluor 488 rabbit anti-goat (Invitrogen) and Alexa Fluor 594 goat anti-rabbit (Invitrogen). Sections were counterstained with DAPI and visualized on a microscope equipped with a digital camera and software. Cell culture and short-hairpin RNA knockdown MCF-7 cells, a generous gift from the lab of Dr. Edward Filardo (Brown University), were cultured in phenol red-free DMEM/F- 12 (Hyclone), 10% fetal bovine serum, 0.1% penicillin/strepto- mycin in a humidified atmosphere (5% CO2/95%air). Retroviral- mediated shRNA knockdown was performed as described in [23]. The oligonucleotides containing targeting sequences (shown in uppercase) for firefly luciferase [short-hairpin luciferase (shLuc)] controls, human Akt1 (shAkt1), and human Btn1a1 (shBtn1a1) PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24432