Figure 6

Involvement of DCC, IN α6β4, Akt, GSK-3β, HSF-1, HSP27, and HSP70 on Ntn-1-induced protection of apoptosis in hypoxic condition. Cells were pretreated with DCC- and IN α6β4 function-blocking antibodies, combination of DCC and IN α6β4 function-blocking antibodies, Akt inhibitor, HSF-1-, HSP27-, HSP70-specific siRNA, and non-targeting control siRNA for 30 min or 24 h prior to hypoxia with Ntn-1 exposure for 72 h. (a) Cells were 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, **P<0.05 versus hypoxia alone, ^#P<0.05 versus combination of hypoxia and Ntn-1. (b) Cells were incubated with 1 μCi of [³ H]-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, **P<0.05 versus hypoxia alone, ^#P<0.05 versus combination of hypoxia and Ntn-1. (c) 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 (top column). Quantitative analysis of the percentage of viable or apoptotic cells by FACS analysis (bottom column). *^,#P<0.05 versus control, **P<0.05 versus hypoxia alone. ROD, Relative Optical Density