Role of NFAT in HIF-1α mRNA expression. (A) RAW264.7 cells were transfected with a NFAT-dependent reporter and prl-cmv-renilla plasmid and treated with the supernatant of apoptotic Jurkat cells (CM_AC-J) with our without 1 μM CsA for 16 hours or with 1 μM CsA alone. Luciferase activity, normalized to Renilla activity, was measured. (B) RAW264.7 cells were treated with CM_AC-J with or without increasing concentrations of CsA for 16 hours. HIF-1α as well as ribosomal 16S protein mRNA were determined, as described in Figure 4. (C) RAW264.7 cells were treated with CM_AC-J with or without 1 μM CsA for 4 hours. Supershift analysis was performed with the addition of the NFATc1 AB. (D) RAW264.7 cells were treated with CM_AC-J with or without 1 μM CsA, 1 μM VPC23019, or 5 μg/mL TGF-β nAB for 4 hours. The TGF-β nAB was preincubated with CM_AC-J for 1 hour at 37°C. Binding of NFAT to a HIF-1α promoter sequence was analyzed by EMSA using a specific 5′-IRD700-labeled oligonucleotide. CsA and VPC23019 were preincubated for 1 hour. Results are representative for 3 individual experiments.