1. The supernatant of apoptotic cells causes transcriptional activation of hypoxia-inducible factor–1α in macrophages via sphingosine-1-phosphate and transforming growth factor-β
by Barbara Herr, Jie Zhou, Christian Werno, Heidi Menrad, Dmitry Namgaladze, Andreas Weigert, Nathalie Dehne, and Bernhard Brüne
Blood
Volume 114(10):
September 3, 2009
©2009 by American Society of Hematology

2. HIF-1α mRNA and protein accumulation in macrophages by CM. (A) RAW264
HIF-1α mRNA and protein accumulation in macrophages by CM. (A) RAW264.7 cells were treated with the supernatant of apoptotic Jurkat cells (CMAC-J) for 4 to 24 hours.
HIF-1α mRNA and protein accumulation in macrophages by CM. (A) RAW264.7 cells were treated with the supernatant of apoptotic Jurkat cells (CMAC-J) for 4 to 24 hours. (B) RAW264.7 cells were treated with CMAC-J, 1 mM DMOG, or the combination of both for 16 hours. Relative expression of HIF-1α and actin was followed by Western blot analysis. Results are representative for 3 individual experiments. (C) RAW264.7 cells were treated with CMAC-J for 1 to 24 hours. (D) RAW264.7 cells were treated with CMAC-J, the supernatant of necrotic (CMNC-J) or viable (CMVC-J) Jurkat cells as well as CMAC-4T1 for 16 hours. HIF-1α as well as ribosomal 16S protein mRNA were determined by qRT-PCR. The ratio of HIF-1α versus ribosomal 16S protein mRNA under control conditions was set to 1. (E) RAW264.7 cells were transfected with the HIF-1α–luciferase promoter plasmid and treated with 10 μg/mL LPS, CMAC-J, or CMAC-MCF7 for 16 hours. Luciferase activity was measured and normalized to protein. Control conditions were set to 1. Data (C-E) are the mean ± SD (n ≥ 3). *Significant alterations compared with controls.
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

3. CM induces HIF-1 activity in RAW264.7 and primary mouse macrophages.
CM induces HIF-1 activity in RAW264.7 and primary mouse macrophages. (A) RAW264.7 cells were transfected with the pGL-3xEPO-HRE plasmid and treated with 100 μM CoCl2 or the supernatant of apoptotic Jurkat cells (CMAC-J) for 16 hours. Control conditions were set to 1. (B-C) RAW264.7 cells or (E-F) primary mouse macrophages were incubated under 1% hypoxia (H) or treated with CMAC-J for 16 hours. Glut-1 (B,E) and VEGF (C,F) as well as ribosomal 16S protein mRNA were determined by qRT-PCR. The ratio of ribosomal 16S protein versus Glut-1 or VEGF mRNA under control conditions was set to 1. Data are the mean ± SD (n ≥ 3). *Significant alterations compared with controls (or otherwise as indicated).
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

4. CM enhances differentiation of EB into CD31-positive cells.
CM enhances differentiation of EB into CD31-positive cells. Plated EB were treated either with (A,C) control medium or the supernatant of macrophages, which were prestimulated for 16 hours with the supernatant of apoptotic Jurkat cells and then incubated for 5 hours with fresh medium (CMPM). CMPM was derived from RAW264.7 (B,D), primary mouse wt macrophages (E), or primary mouse HIF-1α−/− macrophages (F), and added to EB for 24 hours. The appearance of CD31-positive cells was monitored by immunofluorescence staining (red). DAPI is stained in blue. Scale bar represents 200 μm (A-B) and 100 μm (C-F). Results are representative for 3 individual experiments. The dotted line indicates the border between the multilayer body and the surrounding monolayer rim of the EB. Original magnifications, ×10 (A-B) and ×40 (C-E). (G) Mean CD31 fluorescence intensity in the monolayer rim of EB was measured, and the ratio of CD31 versus DAPI under control conditions was set to 1. Data are the mean ± SD (n ≥ 3). *Significant alterations compared with controls (or otherwise as indicated).
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Role of S1P in HIF-1α mRNA induction.
Role of S1P in HIF-1α mRNA induction. (A) RAW264.7 cells were treated with the supernatant of apoptotic Jurkat cells (CMAC-J) for 16 hours in the presence or absence of 1 μM VPC23019 or 100 nM JTE-013. VPC23019 and JTE-013 were preincubated for 1 hour. (B) RAW264.7 cells were treated with the supernatant of apoptotic MCF-7 cells (CMAC-MCF7) or the supernatant of apoptotic MCF-7 cells with a knockdown of SK2 (CMAC-SK2kd), with or without the further addition of 500 nM S1P or 1 μM SEW2871 for 16 hours. HIF-1α as well as ribosomal 16S protein mRNA were determined by qRT-PCR. The ratio of HIF-1α versus ribosomal 16S protein mRNA under control conditions was set to 1. Data are the mean ± SD (n ≥ 3). *Significant alterations compared with controls (or otherwise as indicated).
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

6. Role of TGF-β in HIF-1α mRNA induction.
Role of TGF-β in HIF-1α mRNA induction. (A) RAW264.7 cells were treated with the supernatant of apoptotic Jurkat cells (CMAC-J) for 30 minutes to 4 hours. (B) RAW264.7 cells were treated with CMAC-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 CMAC-J or with denatured CMAC-J (CMAC-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 CMAC-J in the presence/absence of 1 or 5 μg/mL TGF-β nAB, with 5 μg/mL control IgG, with CMAC-J 100°C, or with 10 ng/mL TGF-β for 16 hours. The nAB and the control IgG were preincubated with CMAC-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 CMAC-J with or without increasing concentrations of a TGF-β nAB for 2 hours. The nAB was preincubated with CMAC-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.
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Role of NFAT in HIF-1α mRNA expression.
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 (CMAC-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 CMAC-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 CMAC-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 CMAC-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 CMAC-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.
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology

8. NFAT induces HIF-1 activity and the HIF-1α promoter.
NFAT induces HIF-1 activity and the HIF-1α promoter. (A) RAW264.7 cells were transfected with the pGL-3xEPO-HRE plasmid, cotransfected with a control plasmid (CP) or an NFAT expression plasmid (NFAT-EP), treated with CMAC-J, or remained as controls for 16 hours. (B) RAW264.7 cells were transfected with the HIF-1α-luciferase promoter plasmid, cotransfected with a control plasmid (CP) or the NFAT-EP, and treated in the absence or presence of CsA (preincubated for 1 hour) with CMAC-J, or remained as controls for 16 hours. Luciferase activity was normalized to protein. Control conditions were set to 1. Data are the mean ± SD (n ≥ 3). *Significant alterations compared with controls (or otherwise as indicated).
Barbara Herr et al. Blood 2009;114:
©2009 by American Society of Hematology