cell line: HCT116R cell type: Colorectal cancer parental or resistant: Parental selumetinib resistance concentration: N/A
Treatment protocol
Cells were treated with fresh normal growth medium (with or without selumetinib as described above) for 24 hours.
Growth protocol
Cells were grown in DMEM (HCT116R, HCT116R_6244R, HKH2, HKH2_6244R, LoVo, LoVo_6244R) or Leibovitz's L-15 (SW480, SW480_6244R, SW620, SW620_6244R) containing 10% FBS, penicillin (100 U mL-1), streptomycin (100 mg mL-1) and 2 mM glutamine. Liebovitz’s L-15 media was additionally supplemented with 0.75 mg mL-1 sodium bicarbonate. Cells were incubated in a humidified incubator at 37°C and 5% (v/v) CO2. For selumetinib-resistant cell lines, cells were grown in these media with added 2 µM selumetinib (HCT116R_6244R and HKH2_6244R), 4 µM selumetinib (LoVo_6244R), or 1 µM selumetinib (SW480_6244R and SW620_6244R).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was prepared from the cell pellets using the Allprep DNA/RNA/miRNA Universal kit (Qiagen, Valencia, CA)
Label
Cy3
Label protocol
Genomic DNA extracted from experimental (cell lines) and normal control female gDNA (Promega, Madison, WI) samples was labeled using CytoSure HT Genomic DNA Labelling Kit (Oxford Gene Technology, Oxfordshire, UK) following the OGT protocol for aCGH analysis with an adjustment to the post purification step. After labelled targets were combined in one tube, an additional 2 μl of water was added and directly proceeded to hybridization.
Cells were treated with fresh normal growth medium (with or without selumetinib as described above) for 24 hours.
Growth protocol
Cells were grown in DMEM (HCT116R, HCT116R_6244R, HKH2, HKH2_6244R, LoVo, LoVo_6244R) or Leibovitz's L-15 (SW480, SW480_6244R, SW620, SW620_6244R) containing 10% FBS, penicillin (100 U mL-1), streptomycin (100 mg mL-1) and 2 mM glutamine. Liebovitz’s L-15 media was additionally supplemented with 0.75 mg mL-1 sodium bicarbonate. Cells were incubated in a humidified incubator at 37°C and 5% (v/v) CO2. For selumetinib-resistant cell lines, cells were grown in these media with added 2 µM selumetinib (HCT116R_6244R and HKH2_6244R), 4 µM selumetinib (LoVo_6244R), or 1 µM selumetinib (SW480_6244R and SW620_6244R).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was prepared from the cell pellets using the Allprep DNA/RNA/miRNA Universal kit (Qiagen, Valencia, CA)
Label
Cy5
Label protocol
Genomic DNA extracted from experimental (cell lines) and normal control female gDNA (Promega, Madison, WI) samples was labeled using CytoSure HT Genomic DNA Labelling Kit (Oxford Gene Technology, Oxfordshire, UK) following the OGT protocol for aCGH analysis with an adjustment to the post purification step. After labelled targets were combined in one tube, an additional 2 μl of water was added and directly proceeded to hybridization.
Hybridization protocol
The hybridization master mix was prepared by mixing 25 μl of Cot-1 (1mg/ml), 26 μl or Agilent 1-x Blocking Agent and 130 μl of Aligent 2x HiRPM Hybridization Buffer. The master mix was mixed with labeled experimental and control DNA and hybridized to the Agilent Human Genome CGH 2x400k Miscoarray (Agilent Technologies, Santa Clara, CA). The arrays were incubated for 40 hours at 65°C in a rotating oven at 20 rpm. After hybridization slides were washed as stated in the protocol.
Scan protocol
All hybridized arrays were scanned on an Agilent G2505C scanner.
Description
Parental cell line HCT116R
Data processing
Images were analized using Agilent Feature Extraction Software (version 10.7.3.1). Nexus Copy Number software v7.0 was used for data processing and visualization. Log2 ratio for each gene was calculated as mean of log2 values of all probes mapped to a particular gene.
