To determine gene expression alterations induced by Nkx2-1 knockdown in TnonMet cells Array analyses were performed on 368T1-control (bc37), 368T1-shNkx2-1 (E1), 394T4-control (bc37), and 394T4-shNkx2-1(E1) cells. The 394T4 samples were run in duplicate using either the Affymetrix GeneChip® WT Sense Target Labeling (A9242, A9244, A9245, A9247) or the Nugene Ovation® Systems Target Preparation kits (A9248 and A9249). Thus, a pairwise comparison between the three control and their corresponding shNkx2-1 datasets was used to determine the potential Nkx2-1 regulated genes
Growth protocol
Tumours were initiated by intratracheal infection of KrasLSL-G12D/+;p53flox/flox mice with a lentiviral vector expressing Cre-recombinase. The MIT Institutional Animal Care and Use Committee approved all animal studies and procedures. Cell lines were created from individual lung tumours and metastases harvested from mice 8-14 months after tumour initiation. Tumours were cut into small pieces and then digested for 30 minutes at 37°C in 2ml of HBSS-free containing Trypsin, Collagenase IV, and Dispase in a 15ml conical tube. Following digestion, 4mL of Quench Solution (L15 media with 400μl of FBS and 15μl of 5mg/ml DNase) was added. Digested tumour samples were then pressed through 40μm cell strainers (BD). Finally, samples were centrifuged at 1,000 r.p.m. for 5 minutes, resuspended in culture media (DMEM, 10% FBS, pen/strep, glutamine), and plated in a 12-well plate. Cells were washed and culture media was changed every day for a week until stable cell lines were formed. In general we were able to derive cell lines from approximately 50% of tumours and metastases without a noticeable difference in the frequency of cell line generation from metastases from different site relative to the primary lung tumours.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Trizol (Invitrogen)
Label
biotin
Label protocol
Affymetrix GeneChip® WT Sense Target Labeling
Hybridization protocol
Performed according to manufacturer's instructions (Affymetrix) for Affymetrix Mouse Exon 1.0 ST arrays.
Data processing was done using Partek Genomics Suite 6.5 software. RMA was used to summarize and normalize the probe data into probe set expression values. The list of utilized probe sets was defined by custom .ps and .mps files. The RMA procedure used included the options adjust for GC content, probe sequence, RMA background correction, Quantile Normalization and median polish probe set summarization. Expression values for probesets were summarized to a single value per gene based on the custom file MoEx-1_0-st-v1._core+miRNA+RfSq+UCSC_.SH.2916.mps. The outlier excluded mean method was used. This procedure excludes probe set values that are more than 3 standard deviations above the mean or more and 2 standard deviations below the mean. probe group file: MoEx-1_0-st-v1.r2.pgf meta-probeset file: MoEx-1_0-st-v1._core+miRNA+RfSq+UCSC_.SH.2916.mps
