1. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) Induces Vascular Permeability Factor (VPF/VEGF) Expression by Cultured Keratinocytes 
Jens Gille, Mona Khalik, Veronika König, Roland Kaufmann 
Journal of Investigative Dermatology 
Volume 111, Issue 6, Pages (December 1998)
DOI: /j x
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
VPF/VEGF protein expression is induced by HGF/SF in keratinocytes. VPF/VEGF protein concentration of supernatants derived from confluent cell cultures. (A) Incubation of primary epidermal keratinocytes (on the left) and dermal fibroblasts (on the right) with media only (C), HGF/SF (H), or TGF-α (T) (each at 100 ng per ml) for 24 h. p < 0.01 for VPF/VEGF synthesis by untreated controlsversus HGF/SF-treated keratinocytes. (B) Incubation of HaCaT cells with media only (□) or with 100 ng HGF/SF per ml for 6, 12, 18, 24, 36, and 48 h (▪) prior to analysis. (C) Incubation of HaCaT cells with increasing concentrations of HGF/SF (5, 10, 50, or 100 ng per ml) for 24 h, or incubation with 100 ng TGF-α per ml for 24 h (lane 6), or media change 24 h prior to extraction (first lane). Values are expressed as ng secreted VPF/VEGF protein per mg total cellular protein (mean ± SD, n = 2). The data presented in each graph are obtained from a single experiment representative of three that were performed revealing equivalent results.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
HGF/SF induces VPF/VEGF mRNA expression in HaCaT cells in a (A) time- and (B) concentration-dependent manner. Northern blot analyses of total RNA (20 μg per lane) extracted from confluent cell cultures. (A) Incubation of HaCaT cells with 100 ng HGF/SF per ml for 2, 6, 16, and 24 h or media change 24 h prior to extraction (first lane). (B) Incubation of HaCaT cells with increasing concentrations of HGF/SF (10, 50, or 100 ng per ml) for 6 h, or incubation with 100 ng TGF-α per ml for 6 h (lane 5) or media change 6 h prior to extraction (first lane). (C) Incubation of primary epidermal keratinocytes with media only (C), HGF/SF (H), or TGF-α (T) (each at 100 ng per ml) for 6 h. The lower panels display an image of the respective ethidium bromide (EtBr)-stained nylon membrane to demonstrate even loading and transfer. Results were confirmed in three independent sets of experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Effect of cycloheximide and genistein on the HGF/SF-induced VPF/VEGF expression in HaCaT cells. (A) Northern blot analyses of total RNA (20 μg per lane) extracted from confluent HaCaT cell cultures. Preincubation with cycloheximide (7.5 μg per ml;lanes 3 and 4) or with solvent (DMSO 0.1%,lanes 1 and 2) for 1 h, followed by stimulation with 100 ng HGF/SF per ml (lanes 2 and 4) or media only for 6 h (lanes 1 and 3). (B) VPF/VEGF protein concentration of supernatants derived from confluent HaCaT cell cultures. Preincubation with genistein (20 μg per ml;□) or with solvent (DMSO 0.1%;▪) for 1 h, followed by stimulation with 100 ng HGF/SF per ml (second panel) or media only for 6 h (Ctrl., first panel). Values are expressed as ng secreted VPF/VEGF protein per mg total cellular protein (mean ± SD, n = 2). The data presented are obtained from a single experiment representative of three that were performed revealing equivalent results. p < 0.01 for +/– genistein in HGF/SF-treated cells.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Effect of HGF/SF stimulation on VPF/VEGF mRNA stability. Northern blot analyses of total RNA (20 μg per lane) extracted from confluent HaCaT cell cultures. Incubation with 100 ng HGF/SF per ml for 1 h (in the center) or 16 h (on the right) (first lane) and 1, 2, and 3 h after addition of actinomycin D (7.5 μg per ml). The lower panels display an image of the respective ethidium bromide (EtBr)-stained nylon membrane. Log-linear plot (on the left) of corrected VPF/VEGF mRNA expression (%)versus time (h) including calculated linear regression lines of the different experimental procedures: prestimulation with HGF/SF for 1 h (•) or 16 h (○). Calculated half-lives on the basis of two independent experiments: 1 h HGF/SF (•), 0.65 ± 0.25 h; 16 h HGF/SF (○), 0.75 ± 0.20 h. Results were confirmed in three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
The HGF/SF-responsive region is localized between bp –88 and –70 of the VPF/VEGF gene promoter. Analysis of a sequential series of 5′-deletional VPF/VEGF promoter-based CAT constructs in A431 cells. Schematic representation of the respective reporter gene constructs on the left, coordinates with respect to the transcription start site in the center, and the relative CAT activities with respect to expression of the pCAT Basic vector in graphic format on the right: □, unstimulated controls (Ctrl.);▪, HGF/SF-treated A431 cells. Values represent the mean ±SD of triplicate assays; the fold increase in CAT activity after HGF/SF treatment is depicted to the right (mean ± SD of three independent triplicate transfections and assays). The transcriptional activation experiments were confirmed in HaCaT keratinocytes with results comparable with those seen in A431 cells (data not shown).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
DNA-binding activity of HaCaT nuclear protein extracts to the –88/–65 bp VPF/VEGF promoter sequence. Representative electromobility shift assays using nuclear extracts of untreated cells (lane 1), TGF-α-treated HaCaT cells (lane 2), and HGF/SF-stimulated HaCaT cells (lane 3) in the presence of excess unlabeled double-stranded Sp1 consensus oligonucleotides. The DNA sequence of the utilized probe is shown at the top; formation of the TGF-α-inducible AP-2-dependent complex is marked by abold arrow; unlabeled Sp1 consensus oligonucleotides were consistently used at a final concentration of 0.35 μM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions