selleck catalog We suggest that these 17 genes that were differentially expressed after both PI3K or mTOR inhibitions and p70S6K inhibition with at least two siRNAs against RPS6KB1 are potential downstream targets of PI3K mTOR p70S6K pathway. Conclusion We identified a number of genes that were differentially expressed due to p70S6K suppression and PI3K mTOR pathway inhibitions suggesting novel downstream targets of PI3K mTOR p70S6K pathway. These include VTCN1 and CDKN2B, whose functional role in breast cancer downstream of this pathway requires further investiga tion. Due to the association of p70S6K overexpression with aggressive disease and poor prognosis of breast can cer patients, the downstream targets of p70S6K may have diagnostic value.
Methods Suppression of RPS6KB1 expression by siRNAs BT 474 and MCF 7 breast Inhibitors,Modulators,Libraries cancer cell lines with Inhibitors,Modulators,Libraries high level amplification and overexpression of RPS6KB1 were transfected with three synthetic siRNAs targeting RPS6KB1 purchased from Ambion. Transfections were performed using the Transfecting Stealth RNA or siRNA into Mammalian Cells Using Lipofectamine 2000 protocol according to the manu facturers recommendations. 150,000 cells per well were plated in 2. 5 ml of culture medium onto a 6 well plate 24 hours before the siRNA transfections. For BT 474 cell line, 7. 5 l of Lipofectamine2000 transfection reagent and 50 pmol of RPS6KB1 siRNA were used. For MCF 7 cell line, the conditions were 7. 5 l of Lipofectamine2000 and 200 pmol of siRNA. After four hours of transfection, the cell culture medium was replaced with fresh medium and the cells were incubated altogether for 72 hours.
The cells from two parallel wells were pooled and the total RNAs of BT 474 and MCF 7 breast cancer cell lines were isolated using Qiagen RNeasy Mini Kit. The quality of the RNA was assessed by 2100 Bioanalyzer to control the integ rity of the Inhibitors,Modulators,Libraries samples before microarray hybridizations. Inhibition Inhibitors,Modulators,Libraries of PI3K mTOR pathway by small molecule inhibitors Five breast cancer cell lines, BT 474, Inhibitors,Modulators,Libraries MCF 7, MDA MB 361, MDA MB 436, and SK BR 3, were treated with 50 M of PI3K inhibitor Ly294002 and 100 nM of mTOR inhibitor rapamycin and the cells were harvested at 24 h and 48 h time points. The cell lines were purchased from ATCC and cultured according to the rec ommended conditions. The RNA was extracted using TRI zol reagent according to the manufacturers instructions.
After the selleck Trizol extraction, the RNAs were purified using Qiagens RNeasy column purification to remove remnants from Trizol extraction. Then, all the RNAs were run by 2100 Bioanalyzer before microarray hybridizations. Protein assays by Western immunoblotting Western immunoblotting was carried out to study the pro tein levels of p70S6K, AKT, mTOR and eIF4G1 after the perturbation of the breast cancer cells with PI3K mTOR pathway inhibitors or RPS6KB1 siRNAs.