HIF-1α accumulation in response to oxLDL. (A) MM6 cells were treated with increasing concentrations of oxLDL for 8 hours. For control reasons, 2 μM CuSO₄, 15 μM EDTA (ethylenediaminetetraacetic acid), and a lysate of RCC4 cells were processed in parallel. (B) MM6 cells were time-dependently stimulated with 200 μg/mL oxLDL or nLDL. (C) MM6 cells were pretreated with 3 mM NAC, 30 μM DPI, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) for 2 hours, followed by stimulation with 200 μg/mL oxLDL for 8 hours. S-nitrosoglutathione (GSNO; 1 mM) was supplied together with oxLDL for 8 hours. (D) Cells were pretreated with 30 μM PD98059 or 30 μM SB203580 for 2 hours, following by stimulation with 200 μM oxLDL for 8 hours. (E) HIF-1α expression was induced by exposing MM6 cells to 200 μg/mL oxLDL for 8 hours. Cycloheximide (CHX) at a final concentration of 10 μg/mL was added for the times indicated (15-60 minutes). (F) Cells were treated with 200 μg/mL oxLDL for 5 hours, and HIF-1α mRNA was followed by RT-PCR analysis as described. All experiments were performed at least 3 times and representative data are shown. HIF-1α protein was detected by Western blot analysis as outlined in “Study design.”