Role of TGF-β in HIF-1α mRNA induction. (A) RAW264.7 cells were treated with the supernatant of apoptotic Jurkat cells (CM_AC-J) for 30 minutes to 4 hours. (B) RAW264.7 cells were treated with CM_AC-J with or without 1 μM VPC23019 for 1 or 2 hours. VPC23019 was preincubated for 1 hour. (C) RAW264.7 cells were treated with CM_AC-J or with denatured CM_AC-J (CM_AC-J 100°C) for 1 or 2 hours. Relative expression of Smad2 and the phosphorylation of Smad2 (P-Smad2) were followed by Western analysis. Results are representative for 3 individual experiments. (D) RAW264.7 cells were treated with CM_AC-J in the presence/absence of 1 or 5 μg/mL TGF-β nAB, with 5 μg/mL control IgG, with CM_AC-J 100°C, or with 10 ng/mL TGF-β for 16 hours. The nAB and the control IgG were preincubated with CM_AC-J for 1 hour at 37°C. HIF-1α as well as ribosomal 16S protein mRNAs were determined by qRT-PCR. For details, see Figure 4. (E) RAW264.7 cells were treated with CM_AC-J with or without increasing concentrations of a TGF-β nAB for 2 hours. The nAB was preincubated with CM_AC-J for 1 hour at 37°C. Relative expression of Smad2 and P-Smad2 was followed by Western analysis. Results are representative for 3 individual experiments.