Extraction of Plasmid DNA

E.coli cells containing plasmid DNA were grown in autoclave sterilized LBmedium (10g bacto-tryptone, 5g yeast extract, 10g NaCl in 1l H₂0) with an appropriate antibiotic, ampicillin (100µg/ml) or kanamycin (30µg/ml), overnight at 37°C. Small-scale preparations (minipreps) were performed by the alkaline lysis method (Birnboim and Doly, 1979). Medium (25ml) culture and large (100ml) culture scale preparations of plasmid DNA were carried out by means of the respective Plasmid Midi- and Maxi-Kit from Qiagen (Hilden), according to the manufacturers’ protocol.

Isolation of genomic DNA from mouse tissue

To genotype mice by PCR, DNA was isolated from ear holes. The tissue was lysed at 55⁰C in 50µl of lysis buffer containing proteinase K (1mg/ml). To inactivate the proteinase K, the digests were incubated at 95⁰C for 5min. Lysates were diluted with water and used for PCR.

For Southern blot analysis, lysates from epidermis, tail, or other organs were extracted with phenol/chloroform. The DNA was precipitated with 2 volumes of ice-cold 100% ethanol and dissolved in H₂O at a final concentration of 1mg/ml. The concentration and purity of the DNA were determined by UV-spectrophotometer.

The polymerase chain reaction (Saiki et al., 1985) was used to genotype littermates and each specific PCR was established according to general rules. A list of primers used for genotyping is presented below.

Met^flox

mfs1: 5“-AGCCTAGTGGAATTCTCTGTAAG -3“

mfas2: 5“-CCAAGTGTCTGACGGCTGTG -3“

Met ^null

Wmet5: 5'-CACTGAGCCCAGAAGAGCTAGTGG-3'

neo1L: 5'-CCTGCGTGCAATCCATCTTGTTCAATG-3'

Cre crenew1:

5’-GAACGCACTGATTTCGACCA-3’

crenew2: 5’-AACCAGCGTTTTCGTTCTGC-3’

Deleter

Deleter1: 5'-CGCCATCCACGCTGTTTTGACC-3'

Deleter2: 5'-CAGCCCGGACCGACGATGAAG-3

K14-cre

K14cres: 5’-CTTGCGAACCTCACTACTCG-3’

K14creas: 5’-AGGGATCTGATCGGGAGTTG-3’

Between 5 and 10µg genomic DNA were digested overnight with 20 U of restriction enzyme. The digested DNA was resolved on a 0.8% agarose gel containing ethidium bromide. To confirm complete digestion of the genomic DNA, gel was exposed to UV-light and photographed. The gel was depurinated in 0.25 M HCl solution for 10 to 15min. Then the gel was rinsed in distilled water and denatured by two 30min incubations with gentle shaking in a solution of 1.5 M NaCl and 0.5 M NaOH. Finally the gel was then rinsed in 10X SSC and blotted overnight using 20X SSC, so as to transfer the DNA onto a nylon membrane (Hybond N+, Amersham-Pharmacia). After transfer, the membrane was crosslinked using UVlight at 120mJ/cm2. Subsequently, the membrane was hybridized with specific radioactive probes. DNA probes (20-50ng) were radioactively labeled with 50µCi γ³²PdCTP (Amersham-Pharmacia) using the ‘Prime-It RmT Random-Primed Labeling Kit’ (Stratagene). The labeled probes were purified over Sephadex-G50 spin columns (Probe Quant G50, Amersham-Pharmacia). Before hybridization, probes were denatured by boiling for 5 min.

The membranes were saturated in 20-25ml hybridization solution (6x SSC, 5x Denhardt's solution, 0.5% SDS, 100µg/ml denatured salmon sperm DNA) at 65⁰C for at least 2 hours in the hybridization oven (Biometra). The denatured probes were then added to the tubes incubating the membranes in prehybridization buffer. Hybridization was carried out at 65⁰C for 16-24 hours. In order to remove the non-specifically bound probe, the following washing steps were carried out in a shaking water bath at 65°C: 2x 15min in 2x SSC, 0.1% SDS, 1x 30min in 0.1x SSC, 0.1% SDS. The membranes were then sealed in plastic bags and exposed to a Biomax MS autoradiographic films (Kodak) at –80°C for overnight or exposed to a Phosphoimager (Fujix, BAS 2000) for several hours. If a membrane should be reused for hybridization with a different probe, the old probe was stripped by boiling the membrane in 1% SDS for 30min.

Culture of murine primary keratinocytes

Newborn mice (0-3 days old), under aseptic conditions, were decapitated, limbs and tail were amputated and then the body washed with 1% iodine solution and 70% ethanol. The skin was peeled off, washed in PBS antibiotic solution (gentamycin, GIBCO), carefully laid flat in a sterile cell culture plate, dermis side down, in dispase medium (defined keratinocyte SFM, GIBCO, penicillin/streptomycin, Sigma, dispase II, Roche) and incubated overnight at 4°C with shaking to separate epidermis from dermis. Afterwards the epidermis was separated from the dermis using forceps, incubated with trypsin for 10min at 37°C with vigorously shaking until the solution became opaque. After centrifugation, pellet was washed two times with medium containing FCS and cells were plated on collagen IV coated dishes. Medium containing growth factors was changed every day.

Eight weeks old mice of the same sex were anaesthetized by intraperitoneal injections of ketamine/xylazine (90mg/kg of ketamine and 10mg/kg of xylazine). They were shaved on the back. Two full-thickness excisional wounds, 5 mm in diameter, were made on either side of the dorsal midline by excising skin and panniculus carnosus as described previously (Werner et al., 1994). The wounds were left undressed after injury. For histological analysis, the complete wounds, including 2 mm of the epithelial margins, were excised and either directly embedded in “Tissue-Tec” without prior fixation or fixed overnight in 4% formaldehyde and embedded in paraffin.

Preparation of paraffin sections

Animals were killed by cervical dislocation; back skin was shaved, samples dissected and fixed with 4% formaldehyde at 4°C overnight. After washing with cold PBS, the skin was dehydrated in an ethanol series: 50%, 70%, 80%, 96%, and 100%. After dehydration, it is necessary to replace the ethanol with an agent miscible with paraffin; therefore, skin was incubated in toluol two times for half an hour. Then the skin was incubated in paraffin (Roti-Plast, Roth, Karlsruhe) overnight at 56⁰C, then embedded into cassettes at room temperature, and the resulting solid molds were used for sectioning. The 10µM sections were spread out onto glass slides (Menzel, Braunschweig). The paraffin sections were stained with hematoxylin and eosin (H&E), as well as used for antibody and TUNEL staining.

Preparation of methacrylate sections

For histological analysis skin was also embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim), which is a cold-polymerizing resin. Therefore, skin was fixed and dehydrated in series of graded alcohols. Then skin was incubated in Technovit 7100/100% Ethanol (1:1) for 4-6h at RT followed by overnight preinfiltration in Technovit 7100. Afterwards, the tissue was incubated in infiltration solution (1g of hardener I/100ml Technovit 7100) for 2h up to 2 days at 4°C. Skin was embedded in infiltration solution/hardener II (15:1), which was degassed shortly in vacuum chamber. After overnight polymerization, the blocks were mounted with Technovit 3040 and stored at RT until sectioning. 4-5µm semithin sections were cut using Microm HM360 (Walldorf), dropped into a warm waterbath for spreading and collected onto slides (Roth, Karlsruhe).

Preparation of frozen sections

Laser capture microdissections were performed from frozen sections. The wound fields were excited, snapfrozen in liquid nitrogen and embedded in “TissueTek” („OCT-Compound^“; Sakura, Zoeterwoude, Nederland). The 8μm sections were cut on a cryostat (Microm HM560, Walldorf) and collected onto membrane slides for laser capture microdissections (Molecular Machines & Industries). The sections were fixed in 70% EtOH for 10s and stained with hematoxylin and eosin by immersion using the following protocol: 10s deionized H₂O, 30s hematoxylin, 10s deionized H₂O, 10s 70% EtOH, 1min Eosin Y (alcoholic), 10s 95% EtOH, 10s and 100% EtOH. LCM was performed using an Arcturus PixCell II apparatus, with a 15mm laser beam, power settings of 50–90mW, and laser pulse duration of 6–7mS. This system is based on laser microdissection pressure catapulting technology. A highpressure laser beam ejects the selected sample and catapults it into an Eppendorf cap used with an inverted microscope. A slide with tissue was placed under the microscope, and the wound epithelium was selected using the computer program. The laser cut out the cells and the captured cells were collected into an Eppendorf tube.

Hematoxylin/eosin (H&E) staining on paraffin sections

Hematoxylin solution was prepared by dissolving 4g of hematoxylin in 25ml 95% ethyl alcohol and 40g/400ml NH₄Al(SO₄)₂ ⋅ 12 H₂O. After one-week exposition to air and light, the solution was filtered and mixed with 100ml of glycerin and 100ml of methyl alcohol. Then the solution was exposed to light until it becomes dark (6-8 weeks). Directly before use, the hematoxylin solution was diluted with an equal volume of distilled water.

For H&E staining slides were dewaxed by three incubations in xylene for 10min. Afterwards, slides were hydrated by 5min washes in a series of ethanol (100%, 95%, 80%, 70%) and washed for 2min in distilled water. Such prepared paraffin section as well as methacrylate sections were stained with hematoxylin for 2min and washed in tap water for 10min to allow differentiation. Afterwards, the slides were stained with eosin (0.25% eosin Y, 0.1 M acetic acid) for 5min. After washing, sections were rapidly dehydrated in ethanol series. Finally the sections were washed three times for 5min in xylene, mounted in “Entellan” (Merck, Darmstadt) and coversliped.

Immunostaining

The skin sections were dewaxed and rehydrateted, antigen retrieval was performed by boiling samples in sodium-citrate buffer (C₆H₇O₇Na, 10μM, pH 6.0). Keratinocytes plated on collagen IV coated coverslips were fixed in 4% formaldehyde in PBS for 10min and then subjected to immunostaining. Unspecific binding of antibodies was blocked by incubation with 10% inactivated horse serum/PBT (HS/PBT) for 1-2h at RT. Afterwards, slides were incubated with the primary antibody diluted in 10% HS/PBT O/N at 4°C with rocking or alternatively, the incubation was performed at room temperature for 1h. The following antibodies were used: antivinculin (Sigma), antiRhoA (Santa Cruz Biotechnology), antipaxillin (BD Transduction Laboratories), antiphosphoMet and antiVASP (Cell Signaling Technology), anti-keratin 6 (Covance, Berkeley, CA, USA), anti-keratin10 (Sigma), anti-phosphohistone H3 (Upstate Biotechnology), and anti-PCNA (Oncogene Science). The sections were washed 4 times with PBT for 10min to remove unbound antibodies, and then the sections were incubated with Cy2 or Cy3conjugated secondary antibodies (diluted in 10% HS/PBT) for 1h at room temperature. Sections were washed extensively. Nuclei were visualized by the DNA specific dyeDAPI or Yopro (Molecular Probes) added to the secondary antibodies solution. Finally, slides were covered with “Immunomount” (Shandon, Frankfurt).

Detection of cell proliferation and apoptosis

To detect keratinocytes proliferation, animals were injected intraperitoneally with 75µg of BrdU (5-Bromo-2'-deoxy-uridine) per gram of body weight. BrdU is a thymidine analog and is incorporated into DNA only in mitotically active cells and can be detected using anti-BrdU antibodies. After 1 hour of chasing time, skin samples were embedded in “TissueTek”, as described for preparation of frozen sections. Sections were postfixed in 4% PFA for 15min at RT and then washed with PBS three times for 10min. DNA was denaturated by incubation in 2.4 M HCl for 30min at 37⁰C. Afterwards sections were washed as above and incubated with 20μg/ml proteinase K (Roche, Mannheim) in PBS at RT for 10min to ensure good penetration of the antibody. Afterwards, sections were blocked and immunohistochemistry was performed as described above.

Extensive DNA degradation occurs very often during early stages of apoptosis. Therefore, apoptosis was detected by terminal deoxynucleotidyle transferase nickend labeling (TUNEL ;(Gavrieli et al., 1992). During the TUNEL assay, blunt ends of double stranded DNA breaks are enzymatically labeled with flourescin. The 3-end labeling of DNA breaks was performed using an ‘In situ Cell Death Detection Kit, Flourescein’ (Roche, Mannheim) with minor modifications. Before the procedure, the specimens were heated at 60°C for 1 hour. After deparaffinization in xylene and rehydration through graded ethanol series, the sections were incubated with 20μg/ml proteinase K (Roche, Mannheim) in PBS at RT for 20min. Then the slides were processed according to manufacturer’s instructions.

In situ hybridization

In situ hybridization of paraffin sections was performed using digoxygenin-labeled (DIG) probes (Roche) according to manufacturer’s instructions. The antisense transcripts of mouse cDNAs were as follows: a 1.4 kb HGF/SF fragment that encompasses the 3’coding sequence, a 0.7 kb HGF/SF fragment that encompasses the 5’coding sequence, a 3.7 kb Met fragment, mouse Limd1: RZPD clone IMAGp952H0930Q, mouse Has3 : RZPD clone IMAGp998G102025Q, mouse Igf2: RZPD clone IMAGp998M161029Q

For in situ hybridisation on slides (SuperFrost Plus, Menzel-Glaeser), samples were dewaxed and rehydrated through 75%, 50%, 25% ethanol, PBS for 5 min each. All procedures were performed at RT. Samples were postfixed in 4% PFA for 20min, bleached with 6% H₂O₂ for 15min, and washed 3 times with PBS for 5min each. Samples were treated with 20mg/ml Proteinase K/PBS for 10min and washed in 2mg/ml glycine /PBS for 2min. Samples were postfixed with 4% PFA/PBS for 10min followed by two PBS washes, 5min each. Samples were incubated in 100mM Tris-Cl (pH. 7.5) for 2min, in 100mM Tris-Ac (100 mM Tris-Cl (pH 7.5) supplemented with 0.25% (C₂H₃O)₂O) for 10min, in 2xSSC twice 5min each and dehydrated through 25%, 50%, 75% and 100% ethanol. Air dried samples were hybridised [(33% formamide, 3.3% Boehringer Blocking Reagent (Roche), 3.3 xSSC (pH 4.5), 6.6% dextrane sulfate (Sigma), 3.3mM EDTA (Merck), 0.07% Tween, 100μg/ml heparin, 100μg/ml tRNA, 1μg/ml DIG or Fluorescein–labeled RNA probe)] at 63⁰C overnight in humidified chamber.

The next day samples were washed twice with solution I (50% formamide, 5xSSC (pH 4.5), 0.1% Tween) at 70⁰C for 30min each, three times with solution II (50% formamide, 2xSSC (pH 4.5), 0.1% Tween) at 65⁰C for 30min each, three times with TBST (150 mM NaCl, 100 mM Tris-Cl (pH 7.5), 2 mM KCl, 0.1% Triton-X100) at RT, 5min each. Samples were blocked in 10% sheep serum (GIBCO BRL) in TBST at RT for 90min. After blocking, samples were incubated with the anti-DIG Fab (Roche) coupled to alkaline phosphatase or anti-Fluorescein Fab (Roche) coupled to alkaline phosphatase (1:1000) at 4°C overnight. On the following day samples were washed with TBST at RT for 8h, NTMT (100mM Tris-Cl, pH 9.5, 100mM NaCl, 50mM MgCl₂ (Merck), 0.1% Tween) twice for 20min each. Alkaline phosphatase was detected in NTMT solution supplemented with 4.5μl/ml NBT/INT (Sigma) and 3.5μl/ml BCIP (Sigma) at RT. Sections were dehydrated and coverslipped with Entellan mounting media (Merck).

Galactosidase staining

Frozen sections were fixed in 0.2% glutaraldehyde/PBS at RT for 10min. Following fixation, samples were washed three times for 15 to 30min in lacZ wash buffer (2mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet-P40, NP-40, in 100mM sodium phosphate, pH 7.3, or PBS). Staining was carried out in 0.5mg/ml Xgal, 5mM potassium ferrocyanide, and 5mM potassium ferricyanide in lacZ wash buffer at 37°C or RT for 30min to overnight, with shaking and protection from light. When the staining was complete, slides were rinsed in PBS before dehydration through a graded ethanol series and coversliped.

Extraction of total protein

Proteins were extracted from cultured keratinocytes. Cells were homogenized in icecold 2x RIPA buffer (100mM TrisHCl, pH 7.4, 300mM NaCl, 2mM EDTA, 1% Na-deoxycholate, 2% NP-40, 2mM sodium orthovanadate and 2mM NaF) in the presences of protease inhibitors cocktail (Roche Diagnostic, Mannheim). Lysates were clarified by centrifugation for 45min at 60,000 rpm and supernatants containing proteins were aliquot, snapfrozen in liquid nitrogen and stored at –80°C. All steps were carried out at 4°C temperature.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

The protein electrophoresis was carried out in polyacrylamide gels under conditions that ensure dissociation of proteins. The concentration of the separating gel depends on size of the protein of interest. The electrophoresis was carried out in a discontinuous buffer containing the nonionic detergent, SDS.

Separating Gel:

Resolving buffer was composed from 0.2% SDS, 4mM Na₄EDTA, and 0.75 M TrisHCl pH 8.9

Stacking gel:

In a flask 0.4ml of 30% acrylamide/0.8% bisacrylamide, 1.5ml of 2x Stacking buffer (0.25 M Tris-HCl, pH 6.7, 4mM EDTA, and 0.2% SDS) and 1.05ml of H₂O was mixed. Prior to pouring 30µl of 10% ammonium persulphate and 2µl of TEMED were added.

Prior to loading the protein samples (40-60µg) were diluted 1:2 with 2x Laemmli SDS sample buffer (2% 2-mercaptoethanol, 0.2 M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 0.004% Bromophenol Blue) and heated 5min at 100°C.

The electrophoresis was performed in 1x running buffer (made up from a 4x stock of 0.2 M Tris-HCl, 1.52 M glycine, 0.4% SDS, 8mM EDTA) for 4-5 hours at 30mA constant current.

Western blotting

Proteins were transferred from the gel onto membranes (nitrocellulose or nylon) by a wettransfer method. After separation of the proteins, the gel and the nitrocellulose or nylon PVDF membrane was prewetted in transfer buffer (25mM Tris, 192mM glycine, 20% methanol and 0.1%SDS) for 5min. In case of PVDF membranes, they were first activated for 15sec in 100% methanol. The transfer was performed at 200mA constant current for 2 hours at 4°C (Biorad, Model 200/2.0). Afterwards, the membranes were washed three times with water and the transfer efficiency was determined by Ponceau staining (2% Ponceau, 1% acetic acid in distilled water) for 2min at room temperature with constant shaking. For further processing, the membranes were destained with water for 20min with agitation.

Immunodetection

Nonspecific binding sites on membranes were blocked for 1 h at RT in blocking solution (5%, w/v), skimmed milk powder in PBS and 0.05% Tween-20. The primary antibodies were diluted in blocking solution and incubated with the membrane for 2-3 hours at RT. Antibodies specific to Erk1/2, phospho Erk1/2, Akt, phospho Akt, phospho Gab1, phospho PAK1/2 (Cell Signaling Technology) were used. After washing in PBT (1x PBS, 0.05% Tween 20) four times for 10min, the horseradish peroxidase-conjugated secondary antibodies diluted in blocking solution were applied for 45min. Then the membranes were washed 4 times for 10min in PBT.

For visualization of immunoreactive bands, the chemiluminescent detecting ECL reagent was used according to manufacturer’s instructions (Amersham Biosciences, Freiburg). Briefly, detection solution was applied to the membrane to cover it evenly. After one-minute incubation, excess solution was drained from the membrane, and the membrane was placed on a flat sheet of Saran Wrap. The edges of the wrap were folded over the backside of the membrane to seal it. The membrane was then exposed to a Kodak Xray film for varying lengths of time.