Fig. 3: Role of cP1P-induced HIF1α in apoptosis and therapeutic potential of UCB-MSCs.

a, b UCB-MSCs were treated with cP1P (1 μM), S1P (1 μM), or P1P (1 μM) for 24 h. a The protein expressions of HIF1α and β-Actin were detected by western blotting. n = 4. b HIF1 activities were assessed by dual luciferase reporter assay. n = 5. c UCB-MSCs were pretreated cP1P (1 μM) prior to hypoxia incubation for 48 h. The mRNA expressions of LDHA and PDK1 in UCB-MSCs were normalized by 18 S rRNA expression levels. n = 4. d NT or HIF1A siRNA was transfected to UCB-MSCs for 24 h prior to cP1P (1 μM) treatment for 48 h. Intracellular lactate levels in UCB-MSCs were measured by using commercial kit. n = 4. e, f NT or HIF1A siRNA-transfected UCB-MSCs pretreated with cP1P (1 μM) for 30 min prior to hypoxia treatment for 72 h. e MitoSox-positive cells were measured by using flow cytometer. n = 6. f The percentages of AnnexinV-positive apoptotic cells were measured by flow cytometer. Annexin V-positive/PI-positive cells and Annexin V-positive/PI-negative cells were considered apoptotic cells. n = 6. g–k Mouse skin flap surgery with vehicle, NT-siRNA-transfected UCB-MSCs or HIF1A siRNA-transfected UCB-MSCs with or without cP1P pretreatment for 24 h prior to transplantation was performed as described in Materials & Methods section. g Necrotic regions of skin flaps at post-injection day 8 were compared with those at post-injection day 0. Representative gross images are shown in upper panel. n = 4–6. h Representative histological images are shown in left panel. Tissue samples at post-injection day 11 were fixed and stained with hematoxylin and eosin. Scale bars are 200 μm (Magnification, × 50 or × 100). C crust, GT granulated tissue, D dermis, Ep epidermis. i, j Tissue slide samples were immunostained with HNA, CD31, or α-SMA-specific antibodies. The percentages of HNA, CD31, or α-SMA-positive cells in high power field (HPF) were analyzed by using Metamorph software. Scale bars are 100 μm. Magnification × 100. n = 6. All gross and immunofluorescence images are representative. k The protein expressions of VEGF, EGF, IL-6, IDO-1, and β-Actin in tissue samples were measured by western blotting. n = 4–5. All blot and immunofluorescence images are representative. Quantitative data are presented as a mean ± S.E.M with scatter plots. * indicates p < 0.05. N.D. indicates not detected