LCC2 (derived from LCC1 with increased small-dosage TAM treatment, estrogen-independent, and TAM-resistant) vs. LCC9 (derived from LCC1 with increased small-dosage fluvestrant treatment, estrogen-independent, and cross-resistance to fluvestrant and TAM)
The LCC cell lines were cultured in DMEM/F12 without phenol red, supplemented with 5% DCC and 1% penicillin/streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 95% ambient air and 5% CO2.
Growth protocol
The LCC0 is derived from MCF-7 presenting estrogen-dependent and tamoxifen-responsive phenotype. LCC1 was established from the estrogen-dependent parent cell line MCF-7 through prolonged withdrawal from potent estrogenic stimuli both in vivo and in vitro. The above in vivo selected cell line was further passaged in ovary-comprised nude mice and re-established in vitro to generate a new cell line, MCF-7/LCC1, which is estrogen-independent, but tamoxifen-responsive as its parent cell line. Furthermore, a new cell hormone-independent, tamoxifen-resistant subline, LCC2 was developed through growth of LCC1 in incrementally increased dosages of tamoxifen in vitro. And a new cell hormone-independent, cross-resistance to tamoxifen and fluvestrant subline, LCC9 was developed through growth of LCC1 in incrementally increased dosage of fluvestrant.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated from the LCC cell lines using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. The extracted genomic DNA was verified by agarose gel electrophoresis, and the concentration was estimated by NanoDrop (Thermo Scientific). Furthermore, BioAnalyzer 2100 DNA Chip was used to ensure the quality, quantity and integrity for the genomic DNA. Subsequently, the genomic DNA was subject to digestion of RNase A (Invitrogen), and then purified by ethanol precipitation for subsequent analyses.
Label
Cy3,Cy5
Label protocol
One cell line sample (for instance, LCC2) and reference (for instance, LCC1) DNA was labelled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences), respectively, using a random priming method with the labelling kit BioPrime® DNA labelling system (Invitrogen), and the reverse labelling for sample and reference DNA was performed as well.
Channel 2
Source name
LCC9 (derived from LCC1 with increased small-dosage fluvestrant treatment, estrogen-independent, and cross-resistance to fluvestrant and TAM) vs. LCC2 (derived from LCC1 with increased small-dosage TAM treatment, estrogen-independent, and TAM-resistant)
The LCC cell lines were cultured in DMEM/F12 without phenol red, supplemented with 5% DCC and 1% penicillin/streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 95% ambient air and 5% CO2.
Growth protocol
The LCC0 is derived from MCF-7 presenting estrogen-dependent and tamoxifen-responsive phenotype. LCC1 was established from the estrogen-dependent parent cell line MCF-7 through prolonged withdrawal from potent estrogenic stimuli both in vivo and in vitro. The above in vivo selected cell line was further passaged in ovary-comprised nude mice and re-established in vitro to generate a new cell line, MCF-7/LCC1, which is estrogen-independent, but tamoxifen-responsive as its parent cell line. Furthermore, a new cell hormone-independent, tamoxifen-resistant subline, LCC2 was developed through growth of LCC1 in incrementally increased dosages of tamoxifen in vitro. And a new cell hormone-independent, cross-resistance to tamoxifen and fluvestrant subline, LCC9 was developed through growth of LCC1 in incrementally increased dosage of fluvestrant.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated from the LCC cell lines using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. The extracted genomic DNA was verified by agarose gel electrophoresis, and the concentration was estimated by NanoDrop (Thermo Scientific). Furthermore, BioAnalyzer 2100 DNA Chip was used to ensure the quality, quantity and integrity for the genomic DNA. Subsequently, the genomic DNA was subject to digestion of RNase A (Invitrogen), and then purified by ethanol precipitation for subsequent analyses.
Label
Cy5,Cy3
Label protocol
One cell line sample (for instance, LCC2) and reference (for instance, LCC1) DNA was labelled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences), respectively, using a random priming method with the labelling kit BioPrime® DNA labelling system (Invitrogen), and the reverse labelling for sample and reference DNA was performed as well.
Hybridization protocol
The mixture of the differentially labelled cell line DNA (22~24 ug) and reference DNA (22~24 ug), together with 230 ul (1 mg/ml) Cot-1 DNA (Invitrogen) for blocking hybridization to repetitive sequences was hybridized to the BAC DNA on the CytoChip array. The hybridization was performed with HS 4800 Pro hybridization station (TECAN).
Scan protocol
The array was imaged in a GenePix 4000 B laser scanner (Axon Instruments). After optimizing exposure time, the array was scanned in Cy3 and Cy5 channels, respectively.
Description
Analysis copy number variations between the LCC cell lines [sample] LCC2 vs. [reference] LCC9 LCC2-LCC9_P40-P25_fused.txt_LOG2RATIO CH1/CH2
Data processing
Two single-channel 16-bit images were combined for analysis using the image-analysis software BlueFuse v3.5 (BlueGnome). Bluefuse Multi software performed analysis of the both hybridizations (of a dyeswap pair) simultaneously and combined both hybridization results in one output file. Thus, in our study, the two dye-swap hybridizations have one raw data file (i.e., *combined_output_fused.txt). In *combined_output_fused.txt, CH1=sample and CH2=reference. Normalized log2 ratio (sample/reference) was calcualted. The thresholds for deletion and amplification were by default. The breakpoints in the genome were determined by the flanking clones that located the boundaries of the aberration regions.
