Figure 1.

Stimulation of starved MCF7 cells with FCS increases OGT level. (a) MCF7 cells were maintained in a Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FCS, 2 mℳℒ-glutamine, 5 IU/ml penicillin and 50 μg/ml streptomycin at 37 °C in a 5% (v/v) CO₂-enriched humidified atmosphere. Cells were stopped at G0/G1 using the FCS-starvation method.¹¹ Cells were FCS-starved for 48 h and then cell cycle was released by FCS addition. FCS-induced cells were collected at the indicated times after FCS addition. Cells were washed with 10 ml of cold phosphate-buffered saline and lysed directly on ice with lysis buffer (10 mℳ Tris/HCl, 150 mℳ NaCl, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulfate (w/v) and proteases inhibitors, pH 7.4). Cell lysates were centrifuged (20 000 g, 10 min, 4 °C), pellets were discarded and supernatants boiled for 10 min in Laemmli buffer. Proteins were separated by 10% SDS–polyacrylamide gel electrophoresis and electroblotted on a nitrocellulose sheet (GE Healthcare, Orsay, France). Equal loading was verified using Ponceau red staining. Membranes were saturated for 45 min with 5% non-fatty acid milk in (TBS)-Tween buffer (15 mℳ Tris/HCl, 140 mℳ NaCl and 0.05% Tween20 (v/v), pH 8.0). Proteins were immunodetected using the following primary antibodies; OGT: rabbit polyclonal TI14, 1/2000 (Sigma-Aldrich, Saint-Quentin Fallavier, France); α-tubulin: mouse monoclonal B-5-1-2, 1/5000 (Santa Cruz Biotechnology, Heidelberg, Germany); Erk2: D-2, 1/5000 (Santa Cruz Biotechnology); phospho-Erk1/2: rabbit polyclonal, 1/1000 (Cell Signaling, Danvers, MA, USA); phospho-AKT: rabbit polyclonal, 1/1000 (Cell Signaling) and AKT: mouse monoclonal, 1/2000 (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4 °C, washed three times (TBS–Tween, 10 min) and incubated with appropriate horseradish peroxidase-labelled secondary antibodies at a dilution of 1/10 000 for 1 h. After three more washes, detection was performed with enhanced chemiluminescence (GE Healthcare). (b) Histograms represent densitometric analyses of western blots (WBs). Results correspond to the mean value ±s.d. of three experiments (*P<0.05, **P<0.01, ***P<0.001 respectively, NS not significant). (c) Starved MCF7 cells were stimulated with FCS with or without the protein synthesis inhibitor cycloheximide (CHX) at a concentration of 15 μg/ml. OGT expression was analyzed by WB and protein loading was verified using alpha-tubulin. (d) FCS-starved and 1 h FCS-stimulated MCF7 cells OGT mRNA levels were determined by real-time PCR. Quantitative reverse transcriptase–PCR: Total RNA was reverse transcribed using random primers and MultiScribe reverse transcriptase (Applied Biosystems, Villebon sur Yvette, France). Real-time PCR analysis was performed by Power SYBR Green (Applied Biosystems) in a MX3005P fluorescence temperature cycler (Stratagene, Paris, France) according to the manufacturer's instructions. Results were normalized with respect to RPLP0 RNAs used as internal control. The primers used are as follows: OGT sense 5′-TGGCTTCAGGAAGGCTATTG-3′ and antisense 5′-CAAGTCTTTTGGATGTTCATATG-3′, and RPLP0 sense 5′-GTGATGTGCAGCTGATCAAGA-3′ and antisense 5′-GATGACCAGCCCAAAGGAGA-3′. Results correspond to the mean value ±s.d. of three experiments (NS not significant). Molecular mass markers are indicated at the left.