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Figure 2
The hypoxia mimetics DFO and CoCl₂ increase endogenous D3 in NCLP6E hepatocytes and SK-N-AS neurons. (A) D3 activity and Western blot analysis of HIF-1α protein in NCLP6E hepatocytes 24 h after exposure to the indicated hypoxia mimetics. Hepatocytes that were exposed to hypoxia (without hypoxia mimetics) are included in the HIF-1α Western blot as a positive control. (B) D3 activity and Western blot analysis of HIF-1α protein in SK-N-AS neurons 6 or 24 h after exposure to DFO or CoCl₂. Values are mean ± SEM of 3 cell plates. *P < 0.05; **P < 0.005. (C) Western blot analysis of HIF-1α protein in cells that increase endogenous D3 expression in response to hypoxia (JEG-3, SK-N-AS neurons, and NCLP6E hepatocytes) and in cells with undetectable endogenous D3 activity (HepG2, HEK-293 cells, and MSTO cells). (D) ChIP analysis of the DIO3 or the SCN3A promoter in DFO-stimulated SK-N-AS cells using antibodies directed against endogenous HIF-1α. Representative experiments are shown and were reproduced.