5 bromodeoxyuridine brdu incorporation assay

Assessment of in vitro growth upon RICTOR blockade was performed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay as described [11] (link), [31] (link). Cancer cells transfected with RICTOR siRNA (and respective controls) were seeded into 96-well plates (2x10³ cells/well). Cells were grown with medium containing 10% FCS. After 24 and 48 hours, the MTT assay was performed, as described elsewhere [31] (link). In addition, effects of RICTOR inhibition on growth of tumor cells in vitro were determined in a cell-counting assay as described [31] (link). Briefly, 10⁵ cells were seeded into 6-well dishes; after 24 and 48 hours, cells were trypsinised and counted. Finally, cell proliferation was monitored by 5′-bromodeoxyuridine (BrdU) incorporation assay (Roche Diagnostics, Mannheim, Germany). Three thousand cells were cultured for 24 and 48 hours in 96-well plates and stained with BrdU as previously described [32] . The percentage of cells exhibiting genomic BrdU incorporation was measured by absorbance at 370 nm with Tecan Infinite200 (Tecan, Männedorf, Switzerland). Percentages were calculated relative to ctrl. si.