Methyl-β-cyclodextrin, sphingomyelinase, cholesterol, chloroform, isopropanol and paraformaldehyde, as well as deuterium oxide (D2O) and all chemicals for western-blot analysis were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Triton X-100 was supplied by Merck (Schuchardt, Darmstadt, Germany), and methanol by Acros Organics (Geel, Belgium).
Human erythroleukemia K562 cells were grown in the culture medium RPMI 1640 (Bio-Whittaker Europe, Verviers, Belgium) supplemented with fœtal calf serum (FCS) (10%) and glutamine (2 mM) (R10 medium) at 37°C and 5 % CO2. The K562 resistant variant cells (K562adr) were a gift from F. Calvo, Université Paris 7, and were grown in the same medium with 0.5 μg/ml adriamycin.
Lipids were extracted as previously described , and then desiccated and resuspended in isopropanol. Total cholesterol and free cholesterol were assayed using a kit (Cholesterol RTU, Biomérieux Craponne, and Boeringher, France) based on cholesterol esterase. This was followed by cholesterol oxidase reaction and colour development measured on an absorption spectrometer at 500 nm. Concentration was determined using a standard solution of 0.5 mg/ml cholesterol. We first checked that the solvent (isopropanol or chloroform) did not have any effect on colour development.
Cholesterol extraction using methyl-β-cyclodextrin
Cells were incubated in RPMI, glutamin 2 mM, containing methyl-β-cyclodextrin 5 mM for 2 hours at 37°C, 5% CO2. Controls were incubated in RPMI, glutamin 2 mM, with or without FCS 10% for 2 hours.
After incubation with methyl-β-cyclodextrin, with RPMI+ FCS or with RPMI (the last being used as a control for RPMI+FCS, considering that methyl-β-cyclodextrin is not used in the medium with FCS), cells were collected, washed and resuspended at 150,000 cells/ml in RPMI, glutamin 2 mM, FCS 10%. Cells were seeded in triplicate on 96-well microplates and incubated at 37°C and 5% CO2. Proliferation was assessed by counting cells with a cell coulter (Beckman Coulter, ZI model) at 24, 48 and 72 hours after seeding.
Caveolin-1 detection: western-blot
Cells were collected in late log-phase and washed twice in PBS 150 mM and were resuspended in loading buffer (urea 4 M, lauryl sulphate 3.8%, glycerol 20%, tris base 75 mM pH 6; β-mercaptoethanol 5%; bromophenol blue 5%) for 5 mn at 95°C. They were centrifuged at 12500 g, 4°C for 10 minutes, and 50 μg of extracted protein were deposited per well on an 8% acrylamide gel. Migration was performed in an electrophoresis buffer (TRIS base 125 mM; glycine 250 mM; lauryl-sulphate 0.1%, pH 8.1) for 1 hour at 125 V with a size marker (Kaleidoscope prestained standards Bio-rad). Protein was transferred on a nitrocellulose membrane for 2 hours at 80 V and 4°C in a transfer buffer (tris-base 48 mM; glycine 39 mM, SDS 0.037%, methanol 20%). Non-specific sites were blocked with 1% milk (Regilait, skimmed milk, Saint-Martin-Belle-Roche, France) in TBS 1X (tris-base 20 mM, NaCl 500 mM, and pH 7.5). The nitrocellulose membrane was incubated for 1 hour at room temperature with 1 μg/ml anti-caveolin 1 rabbit antibody (Tebu, France) and washed three times for 10 minutes with TBS before incubation for 1 hour at room temperature with goat anti-rabbit and conjugate secondary antibody (1:3000). After three washes in TBS for 10 minutes, staining was developed in a phosphatase alkaline buffer (NaHCO3 0.1 M; MgCl2, 6H2O 1 mM) with tetrazolium nitro-blue 3% in N,N-dimethylformamide 70% and bromo-4-chloro-3-indolylphosphate p-toluidine 1% in N,N-dimethylformamide 70% for 30 minutes at room temperature in the dark.
Cells were collected at the end of the log-phase, 4 days after seeding, when ML were at a maximum, and washed twice with 1 ml PBS 150 mM at 4°C. They were resuspended in paraformaldehyde (PFA) 3% in PBS 150 mM for 30 minutes at 4°C and washed twice with 1 ml PBS 150 mM at 4°C to be resuspended in triton X-100 1% in PBS 150 mM at 4°C for 30 minutes. Cells or triton-insoluble fractions were collected and washed for NMR experiments at 25°C.
After triton treatment, cells were collected, washed with PBS 150 mM and treated for 35 mn at 37°C with 0.5 units of sphingomyelinase. Cells were washed as described for NMR experiments at 25°C.
Cells were washed twice in 1 ml PBS 150 mM, twice in PBS/D2O, centrifuged at 250 g and counted. Then, 107 cells were resuspended in 400 μl PBS/D2O before transfer to a 5-mm Shigemi NMR tube. Experiments were performed without rotation, and the ?analysed cell pellet was maintained in the coil volume in the Shigemi NMR tube.
The NMR proton spectra of whole cells were obtained at 25°C on a Unity Inova spectrometer (Varian, France) working at 500 MHz. One-dimensional runs were performed by accumulating 128 transients of 90° pulse with 2 s relaxation time. The signal from the residual water was suppressed by the presaturation technique, by using 0.03 mW irradiation for 2 s. Acquisition time was 1.34 s on 16K data points, corresponding to a spectral width of 6 kHz. The Fourier transform was applied without zero-filling using an exponential window multiplication function corresponding to 1 Hz line broadening. The resonances were integrated after automatic baseline correction. Two-dimensional COSY runs were performed with 2K data points in the F2 direction and 256 data points in the F1 direction. The two-dimensional Fourier transformation was applied after zero filling to 512 data points in the F1 direction with a sine-bell function in both directions. Each run consisted of a one-dimensional acquisition and a two-dimensional COSY spectrum. Peak assignments were based on data from the literature. The peak areas were measured by manual integration with the Bruker WINNMR software using a manual tangential baseline correction for each peak, and the assigned peak areas were normalized to the creatine peak area. The following resonances were integrated: methyl group (CH3 at 0.9 ppm), methylene group (CH2 at 1.3 ppm), choline N-trimethyl group (N+(CH3)3 at 3.2 ppm) and creatine (CH3 at 3.05 ppm). The values obtained for the different treatments of the two cells lines were compared by ANOVA analysis followed by a Student-Neumann-Keuls test for group-to-group comparison. P < 0.05 was considered as a significant value.
NMR proton spectra of PFA or triton- or SMase-treated cells were acquired at 500 MHz and 25°C on a Bruker Advance DMX500 spectrometer (Bruker, Wissembourg, France). The runs consisted of a 1D acquisition with similar parameters as the previous 1D spectra, and the spectrum analysis was performed as previously with Bruker WINNMR software.
The possible contribution of lactate to the 1.3 ppm signal was eliminated by analysing 2D Cosy spectra in whole cells, which resolved the resonances of lactate from fatty acid chains. In fixed cells, we calculated the ratio of the double-bond signal (CH = CH at 5.4 ppm) to CH2 peak area. As both groups belong to fatty acyl chains, this ratio remained constant since lactate did not significantly contribute to the increase of the CH2 signal .