MCF-7 cells were stably transfected with pIRES-TGFBR1*6A-HA-FLAG. Clones were picked up and SA5 refers to the clone name. The samples were run in triplicate to collect RNA from 3 separate batches of cells and thus named SA5-1, SA5-2, or SA5-3. “Untreated” refers to the samples that were grown in complete media alone with no exogenously added growth factors.
Treatment protocol
MCF-7 cells stably transfected with pIRES-TGFBR1*6A-HA-FLAG were plated and grown until 80% confluent. The cells were serum starved overnight then refed with complete media for 18 hr before RNA collection.
Growth protocol
MCF-7 cells were cultured in RPMI 1640 (Invitrogen Corp., Grand Island, NY) supplemented with 10% Heat Inactivated FBS (Hyclone, Logan, Utah), 2 mM L-glutamine (Invitrogen Corp., Grand Island, NY), non-essential amino acids, 1,000 units/ml penicillin, 10,000 μg/ml streptomycin, 0.006 mg/ml human recombinant insulin (Sigma, St. Louis, MO), and 0.5 mcg/ml amphotericin B (Biologos Inc., Montgomery, AL).
Extracted molecule
total RNA
Extraction protocol
Qiagen RNA easy kit
Label
biotin
Label protocol
Affymetrix
Hybridization protocol
Affymetrix
Scan protocol
The array was read on the Affymetrix GeneChip Scanner 3000.
Description
All protocols were carried out according to the Affymetrix protocols.
Data processing
The microarray data consists of 54,675 probe sets and were normalized using RMA algorithm. Once normalized, genes that were both up- or down-regulated 1.5 fold in *6A cells (SA5) over *9A cells (WB1), and had a p-value of <0.01 using t-test, were uploaded into the Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA). The Ingenuity Pathway Analysis software sorts the genes into their appropriate signaling pathways
