Supplemental material and methods

Generation of transgenic LPXN mice

The open reading frame of human LPXN (position 92-1254, NM_004811) was amplified from cDNA of prostate carcinoma PC-3 cells, sequenced bi-directionally and cloned into the pBluescript(SK) vector. A 6x c-myc-tag was cut out of pCS2-3´mt (kindly provided by U. Strähle, Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Germany) using SpeI and XbaI and cloned into pBS-hLPXN to generate pBS-hLPXN-cmyc. The minimal rat probasin promoter (-426 to +28bp), which is initially regulated by androgens and restricts expression to prostate epithelial cells of the dorsolateral and ventral lobes (Greenberg et al., 1995), was amplified from rat genomic DNA, sequenced bi-directionally and cloned into pBS-hLPXN-cmyc. The 3´UTR fragment of hLPXN (596 bp, position 1257-1831) was amplified from cDNA of Daudi cells, fully sequenced and cloned into pBS-rPB-hLPXN-c-myc. To generate transgenic mice, the 2.56 kb long vector-free DNA fragment was gel-purified and injected into fertilised mouse oocytes and implanted into pseudopregnant FVB mice. Successful transgene integration and transmission was monitored by PCR on mouse tail DNA.

Plasmids

The open reading frame of human LPXN (position 92-1254, NM_004811) was amplified from cDNA of prostate carcinoma PC-3 cells, sequenced bi-directionally and cloned in frame into the vectors pTriEx 1.1 Neo (for expression of LPXN-His), pCMV-Myc (for expression of Myc-LPXN) and in pEGFP-C1 (for expression of EGFP-LPXN; Kaulfuss et al., 2008).

Transactivation assay

NIH/3T3 cells were maintained in DMEM (Dulbecco´s modified Eagle´s medium, PAN-System) added with 10% FCS and 1.2% penicillin/streptomycine.

The reporter gene assay used to analyse the transactivational function of the transgenic LPXN was performed as described previously (Kaulfuss et al., 2008). Briefly, NIH/3T3 cells were transfected with an expression vector cocktail containing 0.1µg pCMV--Gal, 1µg rPB-LPXN-cmyc or as a control with 1µg pTriEx-LPXN together with 1.0µg MMTV-luc reporter plasmid and 1µg pSV-AR. Transfection efficiency was determined using the pCMV--Gal vector.

Adhesion cDNA array

PC-3 cells were transiently transfected with siRNA against LPXN and as control against the firefly luciferase gene. After 72h total RNA was isolated using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer´s instructions. 3µg RNA per sample was transcribed and radioactively labeled using the AmpoLabelling-LPR Kit and hybridised to two identical cell adhesion array membranes (both SABiosciences, Frederick, USA) overnight at 65°C following the manufacturer´s instructions. After membrane washing the signals were displayed using a Molecular Imager FX and analysed by using the Quantitiy One Software (Bio-Rad, Hercules, CA, USA).