Human multiple myeloma cell lines LP1, RPMI8226, U266 and MM.1S were cultured in RPMI 1640 containing L-Glutamine (Invitrogen, Cergy Pontoise, France), 10% fetal bovine serum (Invitrogen, Cergy Pontoise, France) and 1% penicillin streptomycin (Invitrogen, Cergy Pontoise, France) at 37°C in humidified 95% air and 5% CO2. LP1, MM.1S and U266 were provided by ATCC-LGC (Molsheim, France). RPMI8226 was provided by ECACC from sigma-Aldrich (Sigma Aldrich, St Louis, USA).
Growth inhibition Assay
The cell survival was assessed by using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay (MTT, Sigma Aldrich, St Louis, USA). Cells were cultured in 96-well plates (Costar, NY, USA) for 72 h in the presence of AS602868 (generously provided by Merck-Serono, Geneva, Switzerland), TPCA-1(Sigma Aldrich, St Louis, USA) alone or with an anti-IGF-1Rmonoclonal antibody (generously provided by Roche, Penzberg, Germany). Cells were then incubated with 100 µg MTT, and the resulting crystals were resuspended in 100 µL of isopropanol/0.1 N HCl (Sigma Aldrich, St Louis, USA). Absorbance was measured at 540–690 nm using a spectrophotometer (Thermo Electron Corporation), and inhibitory concentrations 50 (IC50) were determined from cell survival curves drawn with Excel (Microsoft).
Fresh human myeloma cell studies
We received 2 mL samples of bone marrow (BM) from patients with multiple myeloma, after having obtained written informed consent. Samples were lysed with lysis buffer for 10 minutes at room temperature. BM cells were washed and suspended in 300 µl phosphate buffer (PBS). Cells were cultured in 24 well plates (Costar, NY, USA) at 37°C with AS602868 (10 µM), anti-IGF-1R (10 µg/mL) or AS602868+anti-IGF-1R. After 24 h, cells were washed with PBS for 5 minutes, centrifuged at 600 g at room temperature and incubated with labelled monoclonal antibodies directed against CD 38, CD 138, CD221(IGF-1R alpha) and CD45 (antibodies Reference in Table S2
) for 15 minutes in the dark at room temperature. Cells were washed with PBS and suspended in 100 µL Annexin buffer with 1 µl Annexin-V FITC and analysed by flow cytometry on a FACS Canto II (BD Biosciences, Erembodegem, Belgium).
Western blot analysis
Cells (1.107) were exposed to 10 µM AS602868 or/and 10 µg/mL anti-IGF-1R for 16 hours. Following treatment, cells were washed with phosphate-buffered saline (PBS) and then solubilised for 30 minutes on ice in lysis buffer (20 mmol/L Tris-Hcl (pH 6.8), 1 mmol/L MgCl2, 2 mmol/L EGTA, 0.5% NP40) with the complete mixture of protease inhibitors (leupeptin, aprotinin, benzamidine, PMSF, TRCK). After incubation, cell debris and nuclei were removed by centrifugation at 12,000 g for 15 minutes at 4°C. Equal amounts of total protein (50 µg per lane) combined with Laemmli buffer were boiled at 95°C for 5 minutes, and then separated on 12% sodium dodecyl sulphate (SDS)-polycrylamide gels followed by electrophoretic transfer to iBlot Gel transfer stacks PVDF or nitrocellulose (Invitrogen). Membranes were blocked for 1 hour at room temperature with phosphate-buffered saline containing 0.1% Tween® 20 (PBS-T) and 5% dried milk and incubated for all night at 4°C with specific antibody diluted with PBS-T and 5% non fat milk. The antibodies used were directed against Bcl-2 (M0887, 1/500, Dako, Denmark), Bcl-XL (sc-634,1/500, Santa cruz, USA), IGF-1 (sc-1422, 1/500, Santa cruz, USA), P21(sc-397, 1/500, Santa cruz, USA), P53 (clone DO7, 1/1000, Dako, Denmark), IkBα(ser32/ser36, IMG-156,1/500 IMGENEX, USA), Bim (2819), Bad (610392, 1/250, BD transduction, USA), IKKβ (2684), IKKβ (2681), phospho-IKBα (S32/S36) (9426), NF-κB p65 (C22B4), phospho-NF-κB p65 (Ser536), 4EBP 1(9644), Phospho 4EBP-1(2855), p70S6k (2708), phospho p70S6k (9209), cyclin E (4129), cyclin A (4656) (1/1000 or 1/2000, Cell Signalling, USA), Akt (IMG-5411-1, 1/250, Imgenex, USA), phospho Akt (IMG-187-A, 1/500, Imgenex, USA)). Membranes were washed with PBS-T 5% dried milk and incubated with a secondary antibody against mice or rabbit immunoglobulin (1/3000, Caltag, USA) for 1 h. Signal was detected by chemoluminescence using ECL western blotting detection system (GE Healthcare, UK). β- actin clone AC-15 (Sigma Aldrich, St Louis, USA) served as internal control for equal loading.
Cell cycle analysis
Cellular DNA content was determined by flow cytometry. After incubation with 10 mg/mL anti-IGF-1R and/or 10 µM AS602868LP1, RPMI8226, MM.1S and U266 cells were washed with PBS and suspended in 800 µL of propidium iodide solution (0.05 mg/mL) in the dark for 1 h at 4°C. Cells were acquired by flow cytometry and cell cycle distribution was determined using Modfit LT 2.0™ 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 
. 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 37°C 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.
Cells (1.106) were cultured at 37°C with 10 µM AS602868 or/and 10 µM TPCA-1 or/and anti- IGF-1R antibody (10 µg/mL). After 48 h, cells were washed with PBS 1× and suspended in 100 µL Annexin buffer with 1 µl 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 comparisons were made with the Student t-test. The minimal level of significance was p<0.05.