■ Array-CGH experiments
Experiments were conducted as previously described on the Institut Curie one megabase resolution BAC-array (Fix et al., 2008; Vincent-Salomon et al., 2008). The RP11-328L16 BAC contains most of the ALK gene. SNP analyses were based on the 100K Affymetrix SNP arrays (GeneChip 50K array Hind and Xba).
■ Knock-down of ALK expression
● Production of viral particles
Day -1: 293T plating
24h prior to transfection, plate 4x106 HEK293T cells /10 cm dish. 293T cells are maintained in DMEM 10% serum and 1% Penicillin/Streptomycin.
Day 0: Transfection
- At least 30 min before the transfection, replace the medium with 9 ml of fresh DMEM medium complemented with 25 µM chloroquine and preheated at 37°C.
For one dish (10 cm dish), prepare the following transfection mix:
5 µg envelope plamid (pVSV-G codes for the VSV-G envelope)
10 µg packaging plasmid psPAX2
10 µg vector plasmid (lentiviral vector pLKO containing the shRNA of interest)
50 µl of CaCl2 2M
- Briefly mix, then add 500 µl of HBS2X, dropwise under agitation by vortexing.
- Wait for 20 min at RT.
- Add the precipitate dropwise in each dish and mix gently by rotating the plate.
Remove medium around 7 hours post-transfection and put 10 ml/dish of fresh preheated medium.
Day 2: Supernatant collecting
Harvest supernatant containing the viral particles and filtrate on 0.45 µm. The clear supernatants can be kept at 4°C for 4-5 weeks.
● Transduction of the lentiviral constructs into neuroblastoma cells
Day -1: Neuroblastoma cells plating
Plate 4x105 neuroblastoma cells per well of 6-wells dishes. CLB-GA and CLB-MA cells are maintained in RPMI 10% serum and 1% Penicillin/Streptomycin. IMR32 and 106C cells are maintained in DMEM 10% serum and 1% Penicillin/Streptomycin.
Day 0: Transduction of the lentiviral constructs
- Supplement 1 ml of viral supernatant with 8µg/mL polybrene
- Remove medium from the cells and replace by the viral supernatant
- Incubate for 7 hours.
Remove the viral preparation and add fresh preheated medium
Day 1: Selection of transduced cells
- Remove medium and add fresh medium containing the appropriate puromycine concentration.
(600 ng/ml for CLB-GA, 800ng/ml for IMR32 and 1 µg/ml for 106C and CLB-MA).
- For colony formation experiments, 3x104 transduced cells were plated at day 0 in 6-wells dishes and stained with crystal violet at day 10.
For proliferation analysis, 2x104 transduced cells were plated at day 0 in 24-wells dishes. The number of living cells was counted at day 4, 7, 10 and 14 using a Vi-cell XR counter (Beckman Coulter).
- Prepare ice-cold lysis buffer (10 mM Tris-HCl [pH=7.4], 150 mM NaCl, 5 mM EDTA, 20 mM NaF, 25 mM β-glycerophosphate, 1 mM Na pyrophosphate, 10% glycerol, 1% NP40, 0.25 % Na deoxycholate, 1 mM Na3VO4, 1x protease inhibitor; Roche).
- Harvest cells and wash them with PBS. Discard the supernatant and resuspend the pellet in lysis buffer. (100 µl of lysis buffer for 20x106 cells)
- Sonicate the lysates (10 seconds - 3 times).
- Centrifuge 30 min. at 13000 rpm at 4°C. Recover the supernatant.
- Determine protein concentration using the BioRad Protein Assay. Cell lysates may be frozen in liquid nitrogen then stored at -80°C.
- Incubate 250 micrograms of protein lysate with 1 µg of polyclonal ALK antibody (Zymed) for 2 hours at 4°C.
- Add G Sepharose beads (previously washed with lysis buffer) for 1 hour at 4°C.
- Pellet the beads by centrifugation and wash the immunoprecipitates bound to protein G Sepharose twice with lysis buffer (without Na deoxycholate).
Resuspend in gel loading buffer and resolved by SDS-PAGE.
■ In vitro kinase assay
- Perform immunoprecipitations as described before until step 7.
- Pellet the beads by centrifugation and wash the immunoprecipitates twice with buffer containing 10 mM Tris-HCl [pH=7.4], 150 mM NaCl, 5 mM EDTA, 20 mM NaF, 25 mM β-glycerophosphate, 1 mM Na pyrophosphate and 10% glycerol.
- Wash the beads with kinase buffer (20 mM HEPES [pH=7.4], 10 mM MnCl2 and 0.5 mM Na3VO4).
- Resuspend the beads in 40 μl of kinase buffer.
- Perform the kinase reactions for 15 min at 20°C after adding [γ-32P ATP] (10 μCi, 6000Ci/mmol, Perkin Elmer).
- Stop the reactions by the addition of an equal volume of gel loading buffer.
- Perform SDS-PAGE on a 8% acrylamide gel.
- Electrotransfer the gel onto Hybond nitrocellulose membranes (Amersham Pharmacia Biotech).
- Detect the kinase reaction products by autoradiography of the membrane.