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Supplementary Materials and Methods: Lentivirus transfection, siRNA and cell proliferation assays: Lentiviruses were produced in 293T cells and breast cancer cell lines infected with lentiviruses were selected using puromycin. siRNA against AKT1 and AKT2 was obtained from Qiagen (Valencia, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine (Life Technologies, Carlsbad, CA, USA) was used for transfecting siRNA and western blotting was done four days after siRNA transfection. MCF-7 and BT-474 cells were maintained in MEM plus 10% fetal calf serum and switched to phenol red-free MEM with 5% charcoal stripped fetal calf serum four days prior to experiments. Cell proliferation assays were performed using Bromodeoxyuridine incorporation-ELISA (Calbiochem/Millipore, San Diego, CA, USA). PI3K/mTOR/AKT inhibitors were purchased from Selleck Chemicals (Boston, MA, USA). Microarray data analyses: Genes that showed statistically insignificant signals in majority of samples were removed from the microarray data, and only those probes that showed statistically significant signal in at least half of the samples of at least one group were retained. The probe level data was then collapsed to gene level data by retaining only the probes, which showed a maximal coefficient of variation across all samples. The data was imported into partek genomics suite for differential expression analysis. ANOVA analysis was performed to identify genes differentially expressed between AKT1KD vs. pLKO, AKT2KD vs. pLKO, and AKT1KD vs. AKT2KD groups. Genes differentially expressed at p value of <0.01 were considered further for pathway analysis using Ingenuity Pathway analysis software (© Ingenuity systems, CA, USA). Description of supplementary files: The following additional data are available in the online version of the manuscript. Table S1: Sequences of primers used in qRT-PCR Table S2: Genes differentially expressed in pLKO (labeled CTR), AKT1KD (labeled AKT1), and AKT2KD (labeled AKT2) with or without E2 treatment for three hours. ER binding pattern to these genes is also indicated. UNTR- vehicle treated controls Table S3: AKT1 and AKT2-specific E2-regulated genes. Basal expression was normalized between cell lines before calculating fold changes in E2-treated condition. Binding pattern of ER, FOXA1, GATA3, p300, CBP, and SRC1,2,3 to these genes is also shown. Table S4: AKT1-AKT2_FOXA1_Comparison gene: FOXA1:E2 dependent genes are preferentially associated with AKT1. The effects of AKT1 or AKT2 knockdown on the expression of 83 FOXA1-dependent E2-regulated genes were examined. Eight of these genes were found to be associated uniquely with AKT1 (Red). E2F Signature: The effect of AKT1 and AKT2 knockdown on the expression of ER:E2F dependent genes associated with resistance to aromatase and PI3K/mTOR inhibitors. Both isoforms had similar effect on the expression of these genes. Figure S1: Ingenuity pathway analysis of AKT1-dependent E2-regulated genes. Three major pathways are shown. Figure S2: Ingenuity pathway analysis of AKT2-dependent E2-regulated genes. Three major pathways are shown. Figure S3: Prognostic value of E2:ER:FOXA1:AKT1 signature in independent breast cancer datasets. Kaplan-Meier curves for recurrence-free or overall survival of patients with breast cancer subclassified based on ER and Progesterone Receptor (PR) expression or tamoxifen treatment. Tumors were split at median to classify as high or low expressers. Table S1: Primers used for qRT-PCR Forward BTG2 TTC CCA GAC CTG CTT CCA GTC TTT KCNK6 TGG AGA ACT GCC CTT ATG GAG CTT RERG TGC ACT GGA GAA GGG AAC ATC ACA RET TCA CAG ATG CAC AAC ACT CCT CCA SIAH2 GCC TGT TGG TGA TTT GGA TGC TGT SLC22A5 TGC TGT GGA AAC CTC CTT GCT ACT SALL4 TGG AAG GAA GTT GGC CAT CGA GAA BETA-ACTIN AAT GRG GCC GAG GAC TTT GAT TGC Reverse ACA AGA TGC AAG AAC ACA CAG CCT GC TGT GCT TTC AAC ACC TCA CCT CCT TAA TGG CTT GCT TGA CAT GCG TGG TAC AGT GCT GAC AAC ACA GCC AGA TCA AGG GAC CAA TAT GGG AAG GCA AAC CAG CCT TTC CCA AGT GCA TTC TGT ACT GGT TCC ACA CAA CAG GGT AGG ATG GCA AGG GAC TTC CTG TAA