Prior to compound stimulation the cells were starved in 5% charcoal/dextran-stripped fetal bovine serum (CDFBS) containing medium. Stimulations included: insulin NPH (Insuman Basal, Sanofi Aventis), insulin glargine (Lantus, Sanofi Aventis), M1 (metabolite of glargine, Sanofi Aventis), M2 (metabolite of glargine, Sanofi Aventis), glulisine (Apidra, Sanofi Aventis), lispro (Humalog, Elly Lilly), Insulin X10 (not marketed, Novo Nordisk), aspart (B28Asp, Novo Nordisk), detemir (Levemir, Novo Nordisk) and IGF1 (Increlex, Ipsen). All insulin analogues were dissolved in their original vehicle solutions [18]. For the in vitro experiments 1000x stock concentrations were prepared. Except for the first exposure experiment (Figure 1C) in which a dose response of 10, 33 and 100 nM was used, all exposures have been performed with a concentration of 10 nM
Growth protocol
MCF7 IRA, MCF7 IRB or MCF7 IGF1R cells (as described in Arch Toxicol. 2014 Apr;88(4):953-66. doi: 10.1007/s00204-014-1201-2. Epub 2014 Jan 25.) were cultured in RPMI supplemented with 5% (v/v) CDFBS (Hyclone) and used for experiments. Cells have been exposed for 1 or 6 hours to 10 nM of the indicated insulin-like molecule. As a control sample a vehicle stimulation was performed that contained everything except the active compound.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using the RNeasy® Plus Mini Kit (Qiagen, Venlo, the Netherlands) and RNA integrity and quality was assessed using the Agilent bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Label
biotin
Label protocol
The Affymetrix 3' IVT-Express Labeling Kit (#901229) was used to synthesize Biotin-labeled cRNA. From each RNA sample 100 ng was used as input for the labeling reactions.
Hybridization protocol
cRNA was denaturized to prepare for the hybridization on the array. The fragmented cRNA with a concentration of 0.0375 μg/μl was finally utilized for the hybridization on the Affymetrix HT HG U133 plus PM. The Affymetrix HWS Kit (#901530) was used for the hybridization, washing and staining of the chips.
Scan protocol
Scanning of the Array Plates was performed using the Affymetrix GeneTitan scanner
Data processing
BRB Array Tools software was used to normalize the CEL data using the Robust Multichip Average (RMA) method. Significantly differentially expressed genes (p-value < 0.001) between the various experimental conditions were identified with an ANOVA test followed by calculation according to Benjamini and Hochberg (Benjamini and Hochberg, 1995).
