1. Volume 84, Issue 6, Pages 1119-1128 (December 2013)
The proto-oncogene c-Fos transcriptionally regulates VEGF production during peritoneal inflammation 
Rusan Catar, Janusz Witowski, Philine Wagner, Isa Annett Schramm, Edyta Kawka, Aurelie Philippe, Duska Dragun, Achim Jörres 
Kidney International 
Volume 84, Issue 6, Pages (December 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Immunocytochemical characterization of human peritoneal mesothelial cells (HPMCs). Cells were stained for calretinin (red), vimentin (green), cytokeratin (green), fibroblast-specific protein-1 (FSP-1; red), and von Willebrand factor (vWF; green). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Positive controls for FSP-1 and vWF were human peritoneal fibroblasts (HPFBs) and human umbilical vein endothelial cells (HUVECs), respectively. Original magnification × 200.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Vascular endothelial growth factor (VEGF) induction in human peritoneal mesothelial cells (HPMCs) stimulated with a combination of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α)—kinetics and concentration-dependent effects. (a) Kinetics of TGF-β1-induced (1ng/ml) and TNF-α-induced (1ng/ml) VEGF secretion alone or in combination. Asterisks represent a significant difference compared with the predictive additive values at each time point (n=5). (b) Effect of TGF-β1 alone or with TNF-α (1ng/ml; n=5) is concentration dependent. (c) Effect of TNF-α alone or with TGF-β1 (1ng/ml; n=3) is concentration dependent. In b and c, cells were stimulated for 48h. Asterisks represent statistically significant differences compared with the predictive additive values. (d) The effect of antibody with agonistic activity toward TNF receptor 1 (TNF-R1) on TGF-β1-induced VEGF release. Cells were treated with TGF-β1 (1ng/ml) for 48h in the presence of either anti-TNF-R1 or irrelevant antibody of the same class. Asterisk represents a significant difference compared with the control antibody at the same dose (n=4). (e) Effect of neutralizing anti-TNF-α or anti-TGF-β1 antibodies on synergistic VEGF release by HPMCs. Cells were incubated with antibodies (all at 1ng/ml) for 48h. Asterisks represent a significant difference compared with cells treated with TGF-β1+TNF-α in the absence of antibodies (n=4).
Kidney International  , DOI: ( /ki )
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4. Figure 3
Effect of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) on vascular endothelial growth factor (VEGF) mRNA expression and stability in human peritoneal mesothelial cells (HPMCs). (a) Cells were treated with TGF-β1 and/or TNF-α (both at 1ng/ml) for the times indicated. VEGF mRNA was quantified with reverse transcription and quantitative PCR (RT–qPCR) in relation to β2-microglobulin mRNA. The data were expressed as a fold change of the control levels in untreated cells (n=3). (b) HPMCs were stimulated for 6h with TGF-β1 (1ng/ml) in the presence or absence of TNF-α (1ng/ml). After that, cells were washed and spiked with actinomycin D (5μg/ml). Total RNA was extracted at the time points indicated and analyzed by RT–qPCR. The inset shows the data for TGF-β1 and TGF-β1+TNF-α that were expressed as a percentage of values at time 0. Results of a representative experiment of two performed are shown. AU, arbitrary unit.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Identification of sequences in the human vascular endothelial growth factor (VEGF) promoter responsive to stimulation with transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). Cells were transiently transfected with VEGF promoter constructs and then stimulated as indicated. Luciferase activity was determined as described in the Materials and Methods. (a) Time effect of TGF-β1 (1ng/ml) on the full-length VEGF promoter activity. (b) Full-length VEGF promoter activity after 24h of stimulation with TGF-β1±TNF-α (both at 1ng/ml). (c) Effect of progressive 5′-deletions of the VEGF promoter on its activity after 24h of stimulation with TGF-β1+TNF-α (both at 1ng/ml). Asterisks indicate a significant difference compared with the unstimulated controls (n=6).
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6. Figure 5
Detection of nuclear complexes in cells treated with transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). Cells were stimulated with TGF-β1±TNF-α at 1ng/ml, and the nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) in all experiments. (a–c) EMSA with consensus oligonucleotide probes for c-Fos. In b and c, cells were stimulated with TGF-β1±TNF-α for 6h. An EMSA in c was performed either in the presence of a 100-fold molar excess of unlabeled vascular endothelial growth factor (VEGF) DNA (resulting in the lack of a shift—a lower arrow) or in the presence of c-Fos-specific antibody (resulting in a supershift—an upper arrow).(d) EMSAs with consensus oligonucleotide probes for Ets-2, AP-4, c-Jun, and SP-1. Lanes are designated as follows: (-) nuclear extract from control unstimulated cells, (+) nuclear extract from cells treated with TGF-β1+TNF-α for 6h, (S) nuclear extract from control cells in the presence of a competitive unlabeled specific probe, and (N) nuclear extract from control cells in the presence of an unlabeled nonspecific probe.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of c-Fos blockade on vascular endothelial growth factor (VEGF) induction by transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α). (a, b) Cells were transiently transfected with either c-Fos small interfering RNA (siRNA) or scrambled (Scramb) siRNA and then stimulated with TGF-β+TNF-α (both at 1ng/ml) for 3h (n=4). (a) c-Fos protein levels were demonstrated by western blotting, and (b) VEGF release was assessed by enzyme-linked immunosorbent assay (ELISA). Lanes in a correspond to groups shown in b; asterisks represent a significant decrease compared with human peritoneal mesothelial cells (HPMCs) exposed to TGF-β1+TNF-α in the absence of siRNA. (c, d) Cells were pretreated with SR for 1h and then stimulated with TGF-β1+TNF-α (both at 1ng/ml) for 3h also in the presence of SR Dimethyl sulfoxide (DMSO) served as a vehicle for SR HPMCs were then assessed for (c) VEGF promoter activity (n=4) and (d) VEGF mRNA expression (n=6). Asterisks represent a significant difference compared with cells treated with TGF-β1+TNF-α in the absence of SR AU, arbitrary unit.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Effect of interleukin-1β (IL-1β) on transforming growth factor-β1 (TGF-β1)-induced vascular endothelial growth factor (VEGF). (a) VEGF release from human peritoneal mesothelial cells (HPMCs) exposed for 48h to increasing concentrations of IL-1β in the presence or absence of TGF-β1 (at 1ng/ml). #Significant increase in response to IL-1β alone compared with the control. Asterisks represent a significant difference between the combined effect of TGF-β1+IL-1β and the calculated additive value (n=3). (b) An electrophoretic mobility shift assay (EMSA) with c-Fos consensus oligonucleotide probes and nuclear extracts from cells pretreated for 1h with or without SR (1μM) and then stimulated with TGF-β1 (1ng/ml)±IL-1β (100pg/ml) for 3h. Results of a representative experiment of three performed. (c, d) HPMCs were treated as above for 6h and then assessed for c-Fos protein by western blotting (c; results of a representative experiment of four performed, lanes correspond to groups in d) or VEGF release (d; n=4; the symbols represent significant differences vs. unstimulated control (*), vs. cells exposed to TGF-β1 alone (§), or vs. cells treated with TGF-β1+IL-1β (#)).
Kidney International  , DOI: ( /ki )
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9. Figure 8
The involvement of c-Fos in vascular endothelial growth factor (VEGF) release by human peritoneal mesothelial cells (HPMCs) during peritonitis. (a) VEGF levels in dialysates drained either from stable peritoneal dialysis (PD) patients after a routine 4-h dwell (n=12) or from patients with peritonitis at first presentation (n=17); horizontal bars represent mean values.(b) The effect of dialysate on VEGF release by HPMCs. Dialysates from four stable and four peritonitis patients were applied to two separate HPMC cultures (20% v/v). After 48h, the supernatants were collected and assayed for VEGF. To estimate the specific HPMC-derived VEGF release, the concentrations of VEGF present in dialysates were subtracted from those detected in postculture supernatants. (c) The dose effect of exemplary PD effluent drained during peritonitis on VEGF mRNA expression in HPMCs. Cells were treated with increasing doses of effluent for 3h (n=6). An asterisk represents a significant difference compared with untreated controls. AU, arbitrary unit.(d) The effect of SR on dialysate-induced VEGF mRNA in HPMCs. Cells were pretreated with or without SR for 1h and then stimulated for 3h with an exemplary PD effluent (25%) drained during peritonitis, in the presence or absence of SR (n=6).
Kidney International  , DOI: ( /ki )
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