DDIT4 regulates mTOR singling and the expression of pulripotency genes in MSC.(A) MSC were pre-treated with CoCl2 (100 μM), rapamycin (10 nM) or vehicle control for 24 hours, serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. CoCl2 and rapamycin were present at all time during serum deprivation and stimulation period. Data shown is representative of three separate experiments. (B–F) Expression of pluripotency genes Oct4, Nanog and Klf4. (B) MSC were treated with 100 μM of CoCl2 for 1 day (D1) or 4 days (D4) and expression of Oct4 and Klf4 was determined by qRT-PCR (n = 5). (C) MSC were transfected with HIF1α or DDIT4 or control siRNA for 24 h and subsequently treated for a further 24 h with CoCl2 (100 μM) prior to qRT-PCR analysis (n = 5). (D) DDIT4 was transiently overexpressed in MSC and the expression of Oct4, Nanog and Klf4 was determined by qRT-PCR (n = 5). (E) MSC stably transfect with shRNA against DDIT4 or negative control were analysed for mTOR substrate (p-S6K) activation and (F) the expression of pluripotency genes Oct4, Nanog and Klf4. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.