cell type: monocultured HMVEC DM control biological replicate: 6
Treatment protocol
To prepare cocultures, inserts were removed from 100 mm polycarbonate Transwell (Corning, Tewksbury, MA) dishes and hydrated in SAEC complete media in a companion 100 mm dish for at least 1 hour. HMVEC were plated at 1,000,000 cells in the bottom of each Transwell dish (growth area: 55 cm2) and allowed to adhere for at least 1 h without the apical chamber insert. Inserts were returned to the Transwell after hydration, and 1,000,000 SAEC were plated onto the Transwell insert (growth area: 44 cm2). Cells were maintained in 15 mL of complete EBM-2 media in the basolateral chamber and 10 mL of complete SAEC media in the apical chamber.
Growth protocol
SAEC were a kind gift from Dr. Tom K. Hei (Columbia University, New York, NY) (Piao et al. 2005). SAEC were cultured in serum free complete SAGM medium supplemented with various growth factors supplied by the manufacturer (Lonza Walkersville, Inc., Walkersville, MD). HMVEC were a kind gift from Dr. Rong Shao (Biomedical Research Institute, Baystate Medical Center/University of Massachusetts, Amherst, Springfield, MA) (Shao and Guo 2004). HMVEC were cultured in endothelial basal medium-2 (EBM-2) (Lonza) and supplemented with 10% fetal bovine serum (Atlanta Biological, Lawrenceville, GA), 100 U/mL penicillin and 10 ug/mL streptomycin (Lonza), 0.01 ug/mL epidermal growth factor (Sigma), and 1 ug/mL hydrocortisone (Sigma). All cells were maintained in an incubator at 37°C with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from SAEC and HMVEC using RNAprotect Cell Reagent and an RNeasy Mini Kit from Qiagen according to the manufacturer’s protocol (Qiagen, Valencia, CA). RNA concentrations were determined using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Total RNA (250 ng) was used for labeling using a QuickAmp labeling kit (Agilent). Extracted RNA was labeled with cyanine (Cy)-3-CTP (PerkinElmer, Waltham, MA) and reference RNA with (Cy)-5-CTP.
To prepare cocultures, inserts were removed from 100 mm polycarbonate Transwell (Corning, Tewksbury, MA) dishes and hydrated in SAEC complete media in a companion 100 mm dish for at least 1 hour. HMVEC were plated at 1,000,000 cells in the bottom of each Transwell dish (growth area: 55 cm2) and allowed to adhere for at least 1 h without the apical chamber insert. Inserts were returned to the Transwell after hydration, and 1,000,000 SAEC were plated onto the Transwell insert (growth area: 44 cm2). Cells were maintained in 15 mL of complete EBM-2 media in the basolateral chamber and 10 mL of complete SAEC media in the apical chamber.
Growth protocol
SAEC were a kind gift from Dr. Tom K. Hei (Columbia University, New York, NY) (Piao et al. 2005). SAEC were cultured in serum free complete SAGM medium supplemented with various growth factors supplied by the manufacturer (Lonza Walkersville, Inc., Walkersville, MD). HMVEC were a kind gift from Dr. Rong Shao (Biomedical Research Institute, Baystate Medical Center/University of Massachusetts, Amherst, Springfield, MA) (Shao and Guo 2004). HMVEC were cultured in endothelial basal medium-2 (EBM-2) (Lonza) and supplemented with 10% fetal bovine serum (Atlanta Biological, Lawrenceville, GA), 100 U/mL penicillin and 10 ug/mL streptomycin (Lonza), 0.01 ug/mL epidermal growth factor (Sigma), and 1 ug/mL hydrocortisone (Sigma). All cells were maintained in an incubator at 37°C with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from SAEC and HMVEC using RNAprotect Cell Reagent and an RNeasy Mini Kit from Qiagen according to the manufacturer’s protocol (Qiagen, Valencia, CA). RNA concentrations were determined using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy5
Label protocol
Total RNA (250 ng) was used for labeling using a QuickAmp labeling kit (Agilent). Extracted RNA was labeled with cyanine (Cy)-3-CTP (PerkinElmer, Waltham, MA) and reference RNA with (Cy)-5-CTP.
Hybridization protocol
Following purification of labeled cRNAs, 825 ng of Cy3- and Cy5-labeled cRNAs were combined and hybridized for 17 h at 65°C in an Agilent hybridization oven.
Scan protocol
Microarrays were then washed and scanned using an Agilent DNA Microarray Scanner.
Description
Gene expression array
Data processing
In vitro microarray data were exported using Feature Extraction v10 as tab-delimited text files after background subtraction, log transformation, and lowess normalization and reported as log or relative expression of sample compared to universal reference. Data were read from each file into R using a custom script (Guo et al. 2012). For each array, values for control spots, spots which were saturated on either channel, spots which were reported by Feature Extraction as non-uniform outliers on either channel, and spots which were not well above background on at least one channel were considered unreliable and/or uninformative and replaced by “NA”. Values were collated into a single table, and probes for which fewer than 10 present values were available were removed. For probes spotted multiple times on the array, values were averaged across replicate probes. Missing data were imputed using the K-means nearest neighbor algorithm as implemented by the impute.knn function in the impute R package from Bioconductor.
