To assess the glycosylation level of the CFTR protein isoform obtained after am

The cells exposed to SNS 032 showed accumulations of cells in G1 phase, consistent with prior reports that showing that SNS 032 induces a cell cycle arrest. The increased percentages of cells in the G1 phases were also observed in HL 60 cells treated with Ivacaftor 価格 Rapamycin. Next, we set out to unravel the molecular mechanism of action of SNS 032.<br><br> On western blot analysis, LDE225 smoothened 拮抗薬 we observed that SNS 032 dose dependently decreased phosphorylation of RNA pol II at Ser2 and Ser5 in KG 1 and HL 60 cells following 6 h of incubation, These are consistent with the previous report, Interestingly, we found that SNS 032 strongly inhibited phosphorylation of mTOR on Ser2448, a marker for mTORC1 activity, as well as phosphorylation of mTOR protein on Ser2481, a marker for the presence of mTORC2 complexes, The ac tivity of mTORC1 and mTORC2 in HL 60 and KG 1 cells was completely inhibited by the treatment with 200 and 400 nM SNS 032 accompanied by slight degradation of protein expression of mTOR, The downregulation of endogenous levels of mTOR protein phosphorylated at Ser2448 was also confirmed in the treated HL 60 cells using ELISA assays, To test the effect of SNS 032 on unrelated signaling pathways, immunoblotting analysis was performed, The addition of the drug did not suppress extracellular signal regulated kinase Thr202 Tyr204 phosphorylation, p38 mitogen activated protein kin ase Thr180 Tyr182 phosphorylation in HL 60 cells, and also did not decrease signal transducer and activa tor of transcription 5 Tyr694 phosphorylation and STAT3 Tyr705 phosphorylation.<br><br> These data emphasize the specificity of SNS 032 against mTOR activity. Moreover, SNS 032 also successfully inhibited phos phorylation of 4E BP1 and p70S6K, the best characterized targets of mTORC1, To test the effect of SNS 032 on mTORC2 complex, we examined activity of SGK downstream of mTORC2 by assessing LY2109761 dissolve 溶解度 the expression of phosphor NDRG1 at Thr346. SNS 032 reduced the phosphorylation of NDRG1 in a dose dependent manner, Consistently, treatment with this compound significantly decreased the level of phosphor Akt, which is directly downstream of mTORC2, but its inhibitory effect on phosphor Akt was modest, To relate the inhibition of activity of mTORC1 mTORC2 with the induction of cell death, we investigated that whether removal of SNS 032 correlates with the recovery from inhibition of phosphor mTOR and PARP cleavage, a marker of apoptosis, Im munoblotting analysis revealed that there was a partial restoration of activity of mTORC1 and mTORC2, as well as PRAP cleavage.<br><br> We next used three kinds of kinase inhibitor LY294002, Rapamycin, and PP242 as positive controls for the inhibition of mTOR pathway. As shown in Figure 4A, LY294002 and PP242 inhibited cell growth of HL 60 cells in a dose dependent fashion. In contrast, Rapamycin slightly suppressed cell proliferation. Immunoblotting analysis showed that Rapamycin decreased phosphor mTOR at Ser2448 and mTORC1 substrates in cluding p70S6K at Thr389 and 4E BP1 at Thr37 46. Whereas, similar to PP242, SNS 032 significantly inhibited phosphorylation of mTOR at both Ser2448 and Ser2481, and also suppressed phosphorylation of all mTORC1 mTORC2 substrates examined, To gether, these data confirm that SNS 032 not only dephosphorylated Ser2 and Ser5 of RNA polymerase II, it also inhibited phosphorylation of mTOR.