AS602868LP1, RPMI8226, MM.1S and U266 cells were washed with PBS and suspended in 800 mL of propidium iodide solution (0.05 mg/mL) in the dark for 1 h at 4uC. Cells were acquired by flow cytometry and cell cycle distribution was determined using Modfit LT 2.0TM software (Veritysoftware Inc, Topsham, USA). Real-time quantitative RT-PCR Total RNA was converted to cDNA using the Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France). Real- time quantitative RT-PCR was performed using SYBR-Green technology in a Lightcycler (Roche, Mannheim, Germany) as previously described [29]. Forward and reverse primer sequences used are detailed in Table S1. Results were analyzed with RelQuant software (Roche). Mitochondrial membrane depolarization Cells were incubated in the presence or absence of AS602868 or/and anti-IGF-1R antibody for 16 h, washed with phosphate- buffered saline (PBS), and incubated for 15 min at 37uC with 50 nM 3,3 dihexyloxacarbocyanine (DIOC6(3)) (Sigma Aldrich, St Louis, USA). Cells were then washed with and resuspended in PBS. DIOC6(3) fluorescence was measured by flow cytometry. Apoptosis assay Cells (1.106) were cultured at 37uC with 10 mM AS602868 or/ and 10 mM TPCA-1 or/and anti- IGF-1R antibody (10 mg/mL). After 48 h, cells were washed with PBS 16 and suspended in 100 mL Annexin buffer with 1 ml Annexin-V FITC (Roche, Germany). After 15 minute incubation at room temperature in the dark, cells were washed and analyzed by flow cytometry (BD Biosciences, Europe, Erembodegem, Belgium). The Annexin-V FITC positive population was considered as the apoptotic fraction in our experiments. Statistical analysis Statistical comparisons were made with the Student t-test. 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