Fig. 5
Critical roles of COX-2 and PGE2 in the enhancement of immunosuppressive properties induced by mTOR inhibition. a,b Mesenchymal stem cells (MSCs) were treated with 100 nM rapamycin (RAPA) for the indicated time. The activation and protein levels of COX-2, mPGES-1, Akt, GSK-3β were measured by Western blot (a, representative data; b, pooled data). c,d MSCs were pretreated with 10 nM or 100 nM rapamycin followed by treating with 10 ng/ml tumor necrosis factor alpha (TNF-α) plus 20 ng/ml interferon gamma (IFN-γ) for 8 h. The protein level of cyclooxygenase-2 (COX-2) was measured by Western blot (c, representative data; d, pooled data). e MSCs were pretreated with 100 nM rapamycin followed by treating with 10 ng/ml TNF-α plus 20 ng/ml IFN-γ for 48 h. Prostaglain-E2 (PGE 2 ) in supernatants was measured by ELISA. f MSCs were pretreated with 100 nM rapamycin followed by coculturing with peripheral blood mononuclear cells (PBMCs) at the indicated ratio in the presence or not of 10 μM NS-398 (a COX-2-specific inhibitor). PBMC proliferation was analyzed by flow cytometry. g Western blot analysis of the COX-2 knockdown efficiency. h MSCs with or without COX-2 knockdown were pretreated with 100 nM rapamycin followed by coculturing with PBMCs at the ratio of 1:50. Flow cytometry showed the PBMC proliferation after 5 days. i,j MSCs were treated with 100 nM rapamycin for the indicated time. The protein level of COX-2 was measured by Western blot (i, representative data; j, pooled data). Data represent mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01. shNC lentivirus carrying scrambled short hairpin (sh)RNA