tissue: breast cancer cell line cell line: ATCC® HTB-19 treatment: CapG overexpression
Treatment protocol
For stable CapG overexpression in BT-20 cells a PCR-amplified cDNA fragment of human CapG comprising the entire coding region supplemented with flanking NotI and BlnI restriction sites, respectively, was digested with the corresponding enzymes (New England biolabs, UK) and cloned into the retroviral S11IN expression vector. S11IN derives from SFβ11 (kindly provided by Christopher Baum, Hannover Medical School, Hannover, Germany). HEK-239 cells were cultured in DMEM supplemented with 10% FCS, 2 mM K-glutamine, penicillin G (100 U/ml) and streptomycin (100 µg/ml). These cells were cotransfected with 70 µg of helper plasmid pHIT60, 7 µg of pczVSV-G envelope and 7 µg of S11IN (control vector) or S11-CapG-IN by using Fugene 6 (Roche, Germany) following the manufacturers’ instructions. Both vectors are based on SF11 with 3’LTR of the spleen focus-forming virus and internal ribosomal entry site (IRES). Twenty-four hours after transfection, the medium was changed to Iscove’s modified medium. Fourty-eight hours after transfection, supernatants were filtered through a 0.45 µm filter and 1 ml virus supernatant and 5 µl Protamin were used to infect BT-20 cells on 6 well plates with 3.5 x 105 cells/well. Forty-eight hours after infection, cells expressing the S11IN or S11-CapG-IN vector were selected by using G418 to a final concentration of 1 mg/ml. BT-20 overexpressing CapG are called BT-20 CapG. For siRNA treatment 5 x 104 cells per well were placed in 6-well plates one day before they were transfected with siRNA. MDA-MB-231 cells were transfected with commercial available CapG specific siRNA (Ambion Inc., USA) using HiPerFect (Qiagen, Germany) according to manufacturers’ instructions. Final siRNA concentration was 15 nM and the sequence is: 5’-GGUGGUGUGGAGUCAGCAU-3’, which binds within position 335-373 of CapG coding region. MDA-MB-231 CapG knock-down cell are called MDA-MB-231 siCapG. CapG knock-down was verified by Western blot analysis
Growth protocol
Cell lines were grown in complete medium consisting of DMEM basic medium with 1 mg/ml glucose supplemented with 10% Fetal Calf Serum (FCS), 1% glutamic acid, 1% MEM vitamins and 0,1% gentamycin at 37°C and 5% CO2. Passaging was performed every 48h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from samples using RNeasy® Mini Kit (Qiagen, Germany) according to the manufactures’ protocol
Label
Cy3
Label protocol
Synthesis of cDNA and subsequent fluorescent labelling of cRNA was performed according to the manufacturers´ protocol (One-Color Microarray-Based Gene Expression Analysis / Low Input Quick Amp Labeling; Agilent Technologies). Briefly, 100 ng of total RNA were converted to cDNA, followed by in vitro transcription and incorporation of Cy3-CTP into nascent cRNA.
Hybridization protocol
After fragmentation labelled cRNA was hybridized to Agilent 4x44k Whole Human Genome Microarrays for 17 h at 65 °C according to the manufacturers´ protocol.
Scan protocol
Microarrays were scanned in extended dynamic range (XDR) mode as described in the manufacturers´ protocol (Scanner G2505B; Agilent Technologies). Signal intensities on 16 bit tiff images were calculated by Feature Extraction software (FE, Vers. 9.5; Agilent Technologies).
Description
Gene expression after CapG overexpression
Data processing
quantile normalization / baseline transformation to the median of all samples (GeneSpring GX, Vers. 11; Agilent Technologies)
