FIG. 2.

Low oxygen activates Notch signaling in MIAMI cells and increases REST and co-REST expression. (A) Relative fold change of Notch mediator (Notch2, Stat6, and Jagged1) mRNA levels after 7 days at 3% compared with 21% O₂ measured by RT-qPCR and normalized to housekeeping genes, EF1α and RPL13a. (B) Western blot analysis: lane 1 shows protein lysates from MIAMI cells grown at 21% O₂, lane 2 cells were grown at 3% O₂. Top panel shows HIF-1α protein, center panel shows NICD protein. (C) mRNA levels of REST and co-REST as measured by RT-qPCR from MIAMI cells grown at high and low oxygen for 7 days. (D) Protein levels of REST and co-REST were measured by western blot from the nuclear fraction of MIAMI cells grown at high and low oxygen for 7 days (top panel). Coomassie blue staining used as loading control (bottom panel). REST, repressor element-1-silencing transcription factor; RT-qPCR, quantitative real-time PCR. *P ≤ 0.05.