Figure 3.

Inhibiting OGT catalytic activity or interfering with its expression hinders FCS-stimulated cell cycle entry. (a) Following FCS starvation, MCF7 cells were incubated with FCS for the indicated time periods with or without the OGT inhibitor Ac-5SGlcNAc at a final concentration of 100 μℳ. Cell lysates were then analyzed by western blot (WB) according to their O-GlcNAc and cyclin D1 content. Equal loading was checked by using a rabbit polyclonal anti-GAPDH (Abcam, Paris, France) at a dilution of 1/5000. (b) Asynchronous MCF7 cells were reverse-transfected with Lipofectamine RNAiMax (Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions using 10 nℳ small interfering RNA targeting OGT²⁵ or a control siRNA (MISSION siRNA universal negative control #1, Sigma). Cell lysates were analyzed by WB according to their cyclin D1, cyclin E, cyclin A, cyclin B1, phospho-Akt, Akt, phospho-Erk1/2, Erk1/2 and tubulin contents. (c) MCF7 cells (2 × 10³) were cultured in 96-wells plates using Dulbecco's modified Eagle's medium with or without 100 μℳ Ac-5SGlcNAc over 5 days. Each day, cell growth was determined using the MTS reagent method (Promega, Madison, WI, USA) according to the manufacturer's directions at 490 nm (n=6). (d) Starved MCF7 cells were reverse-transfected with Lipofectamine using siRNA targeting OGT or a control siRNA as described in b. 24 h later, cells were FCS-deprived for 48 h and then stimulated for the indicated time periods. Cell lysates were analyzed by WB using anti-OGT, anti-O-GlcNAc, anti-β-catenin, anti-phospho-Akt, anti-Akt, anti-phospho-Erk1/2, anti-Erk1/2, anti-cyclin D1 and anti-tubulin antibodies. (e) Starved MCF7 cells were stimulated with FCS for the indicated time periods in conjunction with 10 nℳ wortmannin, an inhibitor of the PI3K pathway. Cell lysates were analyzed by WB according to their OGT and cyclin D1 content. Activation or inhibition of the PI3K pathway was assessed using an anti-P-Akt antibody. Equal loading was checked by using an anti-tubulin antibody. (f) HEK293T cells were cultured under the same conditions as described for MCF7 and HeLa cells (Figure 1). Following OGT silencing (see above for details), HEK293T cells were transfected with β-galactosidase, TOP Flash, FOP Flash and β-catenin2-XFlag vector or an empty vector by the Lipofectamine2000 reagent (Figure 2b) for 24 h to perform TOP/FOP Flash reporter assay. Histogram represents the relative luciferase activity (RLA). Results correspond to the mean value ±s.d. of three experiments (*P<0.05, ***P<0.001, respectively; NS not significant). OGT and β-catenin expression corresponding to the luciferase activity assay were measured by WB. GAPDH was used to attest equal loading.