Gsk591

For heavy methyl stable-isotope labeling by amino acid in cell culture (hM-SILAC), Roswell Park Memorial Institute (RPMI) medium 1640 lacking l-methionine (Gibco) was supplemented with 10% dialyzed fetal bovine serum (Gibco), 1% penicillin/streptomycin and 0.1mM either l-methionine or l-methionine-methyl-¹³CD₃, DMEM (Gibco) lacking l-methionine, l-arginine, and l-lysine was supplemented with 10% dialyzed fetal bovine serum (Gibco), 1% penicillin/streptomycin, 0.398 mM l-arginine, 0.798 mM l-lysine, and 0.2 mM l-methionine-methyl-¹³CD₃. Otherwise, cells were cultured in the medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Jurkat T cells were cultured in the RPMI medium 1640, and HeLa cells in the DMEM. All the cells were grown at 37 °C in a humidified atmosphere with 5% CO₂. For hM-SILAC, cells were grown for seven cell doublings.
The HeLa cells treated with small molecules or stimulated by heat shock were not metabolically labeled. For small-molecule treatment, GSK591 and MS023 (Selleck) were used to treat HeLa cells at 5 μM and 10 μM for 48 h, respectively. To induce SG assembly, HeLa cells were treated with 200 μM NaAsO₂ or heated at 43 °C for 1 h. For SG disassembly, the medium containing sodium arsenite was removed and the cells were incubated in the fresh medium for 1 h.