Figure 1
Effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis. (a) Cells were exposed to hypoxia for 0–72 h and analyzed for their viability by MTT assay. The values are reported as a mean±S.E. of three independent experiments with triplicate dishes. *, #P<0.05 versus control. (b) Cells were exposed to hypoxia for 0–72 h and pulsed with 1 μCi of [3H]-thymidine for 1 h prior to counting. The values are reported as a mean±S.E. of three independent experiments with triplicate dishes. *, #P<0.05 versus control. (c) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min before being exposed to hypoxia for 72 h, and analyzed for their viability by MTT assay. The values are reported as mean±S.E. of three independent experiments with triplicate dishes. *, #P<0.05 versus control, **P<0.05 versus hypoxia alone. (d) Cells were pretreated with Ntn-1 for 30 min before being exposed to hypoxia for 72 h, and then pulsed with 1 μCi of [3H]-thymidine for 1 h prior to counting. The values are reported as mean±S.E. of three independent experiments with triplicate dishes. *, #P<0.05 versus control, **P<0.05 versus hypoxia alone. (e and f) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min before being exposed to hypoxia for 72 h, and the number of cells and viability was determined using a Countess automated cell counter. The values represent mean±S.E. of five independent experiments with triplicate dishes. *, #P<0.05 versus control, **P<0.05 versus hypoxia alone. (g) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min before being exposed to hypoxia for 72 h. The cells were washed with PBS, stained, and analyzed by flow cytometry. Survival and apoptosis was measured using annexin V/PI staining and flow cytometry. Annexin V/PI double negative cells (Q3) were considered alive, annexin V positive/PI-negative cells (Q4) were considered early apoptotic, and annexin V/PI double-positive cells (Q2) were considered late apoptotic. One representative experiment out of three is shown (left column). Quantitative analysis of the percentage of viable or apoptotic cells by FACS analysis (right column). *, #P<0.05 versus control, **, ##P<0.05 versus hypoxia alone. (h) Cells were exposed to hypoxia for 0–72 h, and Bax, Bcl-2, caspase-9, and caspase-3 were detected by western blot. Each example shown is representative of five experiments. (i) Cells were pretreated with Ntn-1 for 30 min before being exposed to hypoxia for 72 h, and Bax, Bcl-2, caspase-9 and caspase-3 were detected by western blot. Each example shown is representative of five experiments. The right part (h and i) depicting the bars denotes the mean±S.E. of five independent experiments for each condition determined from densitometry relative to β-actin. *, #P<0.05 versus control, **P<0.05 versus hypoxia alone. ROD, relative optical density
