pUHG172-1neo (rtTA) was kindly provided by H. Bujard (Heidelberg, Germany). In order to yield pBIEGFP, EGFP was cloned from pEGFP-C3 into pBI-4 (kindly provided by H. Bujard (Heidelberg, Germany) as an NheI/XbaI fragment. ΔAP-2γ was created by deleting 474 bp (158 aa of the NH-terminus) of the 5'-portion of the murine AP-2γ cDNA. Upon introduction of a start codon using a NotI/NaeI-flanked oligo, ΔAP-2γ was cloned into pBIEGFP using NotI/Sal I restriction sites to generate pBIEGFPΔAP-2γ. The BMP-4 Firefly luciferase construct was kindly provided by M. Moser, (Martinsried, Germany) and the CMV-Renilla luciferase plasmid was obtained from Promega (Mannheim, Germany).
Cell Lines and Cell Culture
The murine breast cancer cell line N202.1A is derived from MMTV-HER-2/neu transgenic mice and was kindly provided by P.-L. Lollini (Bologna, Italy) . N202.1A cells were grown in DMEM-Glutamax supplemented with 20% fetal calf serum and penicillin/streptomycin (Invitrogen, Karlsruhe, Germany). Cells were kept under standard conditions using a cell culture type incubator at 37°C under 7.5% CO2. Functional assays were performed 96 h after addition of doxycycline to the cell culture media (2 μg/ml, BD Biosciences, Heidelberg, Germany).
Transient and stable Transfection
For stable transfection N202.1A cells were transfected using 39 μg plasmid DNA (ratio plasmid - resistance gene 10: 1) using 107.5 μl Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) per 10 cm culture dish according to the manufacturer's protocol. N202.1A cells were selected at 850 μg/ml G418 (Calbiochem, Darmstadt, Germany) for the pUHG172-1neo and 200 μg/ml Hygromycin B (BD Biosciences, Heidelberg, Germany) for pBIEGFPΔAP-2γ and pBIEGFP. For BMP-4 luciferase assays N202.1A cells were transfected with 900 ng plasmid (870 ng BMP-4 Firefly luc +30 ng CMV-Renilla luc) and 1.75 μl Lipofectamine 2000 (Invitrogen, Mannheim, Germany) per 24 well culture dish according to the manufacturer's protocol.
Microscopy and Image Processing
Cells were visualized using a Leica-DM-IRB microscope (Bensheim, Germany), fitted to a Microfire digital camera (Optronics, Goleta, CA, USA) and image processing was performed applying Adobe Photoshop and Illustrator software.
HCT116 cells were transfected with equal amounts of AP-2α- and ΔAP-2γ - expression plasmids. Cells were lysed after 48 hours with non-denaturing lysis buffer. Co-IP was performed with 20 μl DYNABEADS®(Invitrogen Cat.no. 199.03D, Invitrogen, Karlsruhe, Germany) und 1,5 μg anti-AP-2α antibody (H79, Santa Cruz, Heidelberg, Germany). 150 μg protein lysate per sample was loaded. Western blot with anti-AP-2γ antibody (6E4/4, Santa Cruz, Heidelberg, Germany) followed.
Western Blot Analysis, Luciferase Assays and Giemsa Staining
Western Blot was performed using the following primary antibodies: AP-2γ (6E4/4, 1:200 Upstate, New York, USA), AP-2α (H79, 1:200 Santa Cruz Heidelberg, Germany), AP-2β (1:1000), AP-2δ (1:1500) and AP-2ε (1:1500), all kindly provided by M. Moser (Martinsried, Germany). The following secondary antibodies were used: goat anti-rabbit-HRP (1:2000 DAKO, Hamburg, Germany) and rabbit anti-mouse HRP (1:1000, DAKO, Hamburg, Germany). Protein lysates from HeLa (cervical carcinoma) N2A (neuroblastoma) cell lines and human keratinocytes or in vitro synthesized proteins, respectively, served as antibody positive controls for the different AP-2 isoforms in Western Blot analysis. BMP-4 promoter luciferase assays were performed using the "Dual Luciferase Reporter Assay Systems Kit" (Promega, Mannheim, Germany) according to the manufacturer's protocol. 48 h upon induction (2 μg/ml doxycycline), N202.1A cells were transfected with BMP-4-Firefly luc, lysed, and luciferase activity was measured at 562 nm (Berthold Technologies, Bad Wildbad, Germany). Transfection efficiencies were normalized using a CMV-driven Renilla Luciferase (CMV-Renilla luc). To quantifiy cell numbers after treatment with chemotherapeutic compounds and irradiation cells were stained with Giemsa. For this purpose cells were washed twice in ice-cold PBS and fixed in methanol for 5 min at room temperature, stained for 5 min in Giemsa solution (Merck, Darmstadt, Germany) and washed gently in H2O. Cells were quantified using ImageJ software (Adriamycin treatment) or three independent fields of visions were counted (irradiation) in each experiment.
Determination of S-phase index was carried out using the "Click-iT™ EdU Imaging Kit" (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Briefly, 96 h after addition of doxycycline (2 μg/ml) N202.1A cells were incubated with 100 μM thymidine analogon EdU for 30 min at 37°C, fixed in 3.7% formaldehyde for 15 min and washed twice in 3% BSA/PBS. Permeabilization was achieved using 0.5% Triton X-100/PBS for 20 min and cells were washed twice with 3% BSA/PBS. Subsequently, the fixed cells were incubated with the reaction cocktail containing Alexa Fluor®594-azide for 30 min and washed with 3% BSA/PBS. Imaging using a Leica DM-IRB (Leica, Bensheim, Germany) microscope was followed by quantification of Edu Alexa Fluor®594-azide staining in three fields of vision (magnification ×100) in three independent experiments. On average, a field of vision contained 218 cells.
Transmission (TEM) - and scanning electron microscopy (SEM)
For TEM-analysis, cells were fixed in 1.25% glutaralaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h and post-fixed in 2% OsO4 in cacodylate. Following dehydration in a graded series of ethanol, the specimens were embedded in a propylene-epon-mixture, mounted as 50 nm thin sections and post-contrasted with 3.5% uranylacetate. TEM analysis was performed using a CM10 microscope (Philips, Eindhoven, Netherland).
For SEM cells were fixed in 2% glutaralaldehyde in 0.1 M sodium cacodylate (pH 7.3) for 20 min, transferred to 0.1% aqueous tannic acid and rinsed with distilled water. All specimens were dehydrated through a graded series of ethanol and critical point-dried from CO2 in 10 cycles using a Balzers CPD 030 (BAL-TEC, Schalksmuehlen, Germany). Dried specimens were mounted on aluminium sample holders and coated with a 2 nm layer of platinum/palladium in a HE 208 sputter coating device (Cressington, Watford, UK). SEM analyis was performed with an XL 30 SFEG (Philips, Eindhoven, Netherlands).
Caspase 3/7 Acitivity and AnnexinVC3.18 staining
Caspase 3/7 activity was quantified using the "Caspase-Glo® 3/7 Assay Kit" (Promega, Mannheim, Germany) according to manufacturer's instructions. Non-induced or cells induced with doxycycline (2 μg/ml) for 96 h were incubated with "Caspase-Glo® 3/7 Reagent" for 3 h followed by measurement of luminescence at 562 nm (Berthold Technologies, Bad Wildbad, Germany). AnnexinVC3.18 staining was carried out using the "AnnexinVC.18 Kit" (Sigma-Aldrich, Munich, Germany). Doxycycline-induced cells were washed 3 times in PBS followed by 3 cycles of binding buffer for 1 min. In the next step cells were incubated with the AnnexinVC3.18 conjugate (AnnexinVC3.18:binding buffer, 1:100) for 10 min at room temperature, washed 3 times in binding buffer and then subjected to fluorescence microscopy. Cells treated for 2.5 h with Staurosporine (2 μM) served as positive controls (Sigma-Aldrich, Munich, Germany).
The irradiation of the N202.1 cells 96 h after addition of doxycyline (2 μg/ml) at 105 Gy was performed using a linear accelerator (Siemens Mevaton MD2, Siemens Medizintechnik, Munich, Germany). The photon energy of 6 MeV at a dose rate of 2 Gy/min was chosen. The field size was set to 20 cm × 20 cm at a SSD (skin to surface distance) of 100 cm. The beam divergence was approx. 11°. The cells were irradiated in a 6-well plate using a RW3-Phantom (PTW) at dose maximum. The photon beam was calibrated according the DIN 6800-2 protocol for a water equivalent energy dose. The RBW-factor for 6 MeV irradiation is almost 1, so the energy dose was equivalent to the biological dose.
RNA isolation and qRT-PCR
Total RNA was isolated from the various clones using Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. 1 μg of DNase-treated (DNA-free™ kit, Ambion, Austin, TX) total RNA was reverse transcribed using RETROscript™ reagents (Ambion, Austin, TX). RNA was heat-denatured for 3' at 85°C and the reaction was incubated at 42°C for 1 hour and 10' at 92°C. Quantitative Real Time PCR (qRT-PCR) reactions were carried out in 96 well plates using SYBRGreen®Master Mix (Applied Biosystems, Foster City, CA), specific primers and 10 ng total RNA converted into cDNA in 10 μl final volume. Fluorescence was measured using an ABI Prism® 7300 (Applied Biosystems, Foster City, CA) detection system according to the manufacturer's instructions. Primers were purchased from QIAGEN (QuantiTect® Primer Assays). Relative quantitations to control cells were performed: first, each Ct value was corrected for the Ctr of the reference gene, GAPDH, and then the Ct of each sample was subtracted from the Ct of control cells (Ct0). The relative amount of template (Q) was therefore calculated as: . All samples were run in triplicates and mean and standard deviation calculated as described in Bookout et al. 2003 . QuantiTect® Primer Assay catalogue numbers are as follows: QT00101297 Mm_Tcfap2c_1_SG QuantiTect Primer Assay (200) (NM_009335, NM_001159696); QT00265524 Mm_Egr3_1_SG QuantiTect Primer Assay (200) (NM_018781); QT00096131 Mm_Ctgf_1_SG QuantiTect Primer Assay (200) (NM_010217); QT01044295 Mm_Sema3b_1_SG QuantiTect Primer Assay (200) (NM_009153); QT00157381 Mm_Nrp1_1_SG QuantiTect Primer Assay (200) (NM_008737); QT01061599 Mm_Gsta3_1_SG QuantiTect Primer Assay (200) (NM_010356); QT00100653 Mm_Gzme_1_SG QuantiTect Primer Assay (200) (NM_010373); QT00134064 Mm_Tnfaip3_1_SG QuantiTect Primer Assay (200) (NM_009397). QT00105483 Mm_Fst_1_SG QuantiTect Primer Assay (200) (NM_008046).
Northern Blot analysis was carried out as described in Jäger et al.  using the murine AP-2γ cDNA as a probe.
Whole genome expression analysis
Microarray analysis of gene expression in response to expression of ΔAP-2γ/EGFP (Δ#7 + Δ#15) or EGFP (Δ#5 + Δ#11) in N202.1A breast cancer clones (2 μg/ml doxycyline for 96 h) was performed using the Illumina BeadChip system (Illumina, Inc, San Diego, CA). We used 500 ng of total RNA to obtain labeled, amplified cRNA for each sample to hybridize the Illumina Ref-8 BeadChips according to manufacturer's instructions (Illumina, Inc, San Diego, CA). Arrays were scanned with an IlluminaBeadArray Reader confocal scanner and data processed and analyzed using IlluminaBeadStudio software (Illumina, Inc, San Diego, CA). Raw Illumina data were rank invariant normalized with BeadStudio software (Illumina, Inc, San Diego, CA), which was also used to assess differential expression between the ΔAP-2γ and control clones, based on three RNA preparations from each clone after subtraction of the background obtained with control clones following doxycyclin treatment. After normalization, genes were filtered by their 'detection' value, which had to be 0.99 (significantly detected), in the three samples. Subsequently, we identified differentially expressed genes using the Illumina custom error model implemented in BeadStudio, which provides an expression difference score ('DiffScore') taking into account background noise and sample variability . We chose a DiffScore threshold of 30, corresponding to a p value of 0.001, with a False Discovery Rate (FDR) lower than 5%. To restrict the analysis to the most regulated genes, an additional filtering criterion was that the average expression fold-change between ΔAP-2γ/EGFP- (Δ#7 or Δ#15) and EGFP- (Δ#5 or Δ#11) expressing clones had to be at least 1.5-fold, which lead to the identification of 139 modulated transcripts (49 decreased and 90 increased). Sample permutation analysis confirmed that under these conditions the FDR was well below 5%.
Gene Set Enrichment Analysis
Gene Set Enrichment Analysis (GSEA) is a computational method used to look for overlaps between the AP-2-driven gene set obtained from the microarray analysis and modulated genes present in the Molecular Signature Database (MSigDB) following chemical and genetic perturbations .
Chromatin immunoprecipitation (ChIP) assays
ChIP was performed using the ChIP-IT™ Kit (Active Motif, Carlsbad, CA) reagents and protocols. Primer pairs were designed on the TFAP2 binding site containing regions (identified by TRANSFAC) using the Primer-BLAST software http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=NcbiHomeAd. The following primers were used: Tnfaip3FW: 5'-CCCCTAACGGAGGCACTCTTCCAC-3'; Tnfaip3RV: 5'-CCGCCTCCTCCAGGTCTTCCTAGCCC-3'; CtgfFW: 5'-AGGAAGTCTC GGGCCTCTTCTCTTTGA-3';CtgfRV: 5'-TCAAGTGGCTGACCACATCATCTGCAC-3'.
PCR was performed using Platinum® Taq DNA Polymerase according to the manufacturer's instructions.