Fig 2: TNFa and IFN? acutely increase the phosphorylation of AKT. a MSCs were stimulated with TNFa and IFN? (10 ng/ml each) for different periods and lysed. Total AKT and phosphorylated AKT (S473) were examined by western blotting. b MSCs were pretreated with LY294002 (LY) or triciribine (TCN) for 1 h and then TNFa and IFN? were added into the medium for 24 h. MSCs were lysed and total protein was extracted. IDO1, total AKT, and phosphorylated AKT (S473) were examined by western blotting. c MSCs treated as in (b), and TSG-6 in the medium were examined by ELISA. d MSCs were infected by constitutive active form Myr-AKT or dominant-negative AKT (DN-AKT) adenovirus two days before TNFa and IFN? stimulation. After TNFa and IFN? treatment for 24 h, MSCs were lysed and total protein was extracted. IDO1, total AKT, and phosphorylated AKT (S473) were examined by western blotting. HA-tagged Myr-AKT or HA-tagged DN-AKT was immunoblotted by antibody against HA. e MSCs treated as in (d), and TSG-6 in the medium were examined by ELISA. f Extracellular acidification rate (ECAR) of hUC-MSCs was monitored when TNFa and IFN? treatment for 1 h. g The basal and (h) maximal glycolysis values in figure (f) were statistically analyzed. (i) Calculation of the glycolysis reserve in figure (f) is subtraction basal glycolysis from maximal glycolysis. Data are presented as mean ± SEM. of triplicates (d–i) ***, p < 0.001

Fig 3: Blocking glycolysis of hUC-MSCs abolishes the expression of cytokines and anti-inflammatory factors. a–e CXCL9, CXCL10, CXCL11, IDO1, and TSG-6 mRNA levels of MSCs were measured. The MSCs were treated with TNFa and IFN? (10 ng/ml each) with or without 2-Deoxy-D-glucose (2-DG) and oligomycin (OM) for 24 h. All sample transcript levels were normalized to the level of ß-actin. MSCs were treated as in (a), then the cells were collected and the IDO1 protein level (f) was examined by western blotting. The TSG-6 protein level (g) in the medium was examined by ELISA. Data are presented as mean ± SEM. of triplicates (a, c) ***, p < 0.001