FIG. 4.

Hypoxic culture condition activates Notch signaling but not HIF proteins. hADMPCs were expanded under normoxic (20% O₂) and hypoxic (5% O₂) conditions. DAPT (1 μM) was added to inhibit Notch signaling. (A) Western blot analysis of intracellular domain of Notch1 (Notch1 ICD) expression. Actin served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (B) Q-PCR analysis of HES1. Each expression value was calculated with the ΔΔCt method using UBE2D2 as an internal control. (C) Western blot analysis of HES1 in nuclear fractions of hADMPCs. Lamin A/C served as the loading control. (D, E) Western blot analysis of HIF-1α (D) and HIF-2α (E). Cobalt chloride (CoCl₂) was added at a concentration of 100 μM to stabilize HIF proteins (positive control). (F) Western blot analysis of phosphorylated Akt (p-Akt) and Akt. Actin served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (G) Western blot analysis of nuclear localization of p65. Lamin A/C served as the loading control. Numbers below blots indicate relative band intensities as determined by ImageJ software. (H) Western blot analysis of phosphorylated p53 (p-p53) and p53. Actin served as the loading control. (I) Activity of p53 was measured by the p53-luciferase reporter assay. Relative luciferase activity was determined from three independent experiments and normalized to pGL4.74 activity.