Cell lines and tissue culture
Synovial tissue samples from patients with RA were obtained immediately after opening the knee joint capsule as previously described [9, 11, 12]. RASF were cultured in DMEM (Sigma-Aldrich, St. Louis, Missouri, USA) + 10 % fetal calf serum (PAN Biotech, Aidenbach, Germany) + penicillin/streptavidin (PAN Biotech). These cells were incubated under humid conditions in a 5 % CO2 incubator at 37 °C.
The human melanoma cell lines Mel Im, A375, Hmb2, HTZ19d, Mel Wei, Mel Ei were maintained in DMEM and Mel Juso in RPMI (Sigma-Aldrich) supplemented with 10 % FCS and P/S. The cells were cultured in an incubator at 37 °C with 8 % CO2.
Isolation of RNA, reverse transcription and quantitative real-time PCR
Total cellular RNA was isolated from cultured cells using the Total RNA Kit (VWR, Darmstadt, Germany) and cDNAs were generated as described before [13]. Real-time quantitative PCR (qRT-PCR) was performed using SYBR Green master mix (QIAGEN) with primer pairs for Robo3A (5′- GAACTTGTTCGCGGACTCTC -3′ and 5′- CCCGTTCTTGTACCACTCA -3′), Robo3B (5′-CCCTCTGGAGCCTCAATCTC-3′ and 5′-CCCCGTTCTTGTACCACTCA -3′), β-actin (5′-CTACGTGGCCCTGGACTTCGAGC-3′ and 5′-GATGGAGCCGCCGATCCACACGG-3′) and MIA (5′-TTCAGGGTGACTACTACGGTCGCCTGGCTGCTCGTCTGGG-3′ and 5′-CCCAGACGAGCAGCCAGGCGACCGTAGTAGTCACCCTGAA-3′). Relative gene expression was normalized to β-actin mRNA levels using the comparative cycle threshold (Ct) method. Specificity of the Robo3 isoform primers was confirmed by sequencing of the PCR products, Robo3A and Robo3B.
Protein isolation and western blot analysis
Protein extracts from primary cells and cell lines were homogenized in 100 or 200 μl RIPA-buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 1 % (w/v) Nonidet® P40; 0.5 % (w/v) Natriumdesoxycholat; 0.1 % (w/v) SDS; Protease inhibitors). Insoluble fragments were removed by centrifugation at 13,000 rpm for 10 min. The supernatant was stored at −20 °C. For western blot, protein lysates were separated on 7.5 % SDS-PAGE gels and blotted onto a PVDF membrane [14]. After blocking with 5 % milk powder/TBS-T (1 %), primary antibodies were applied (anti-Robo3B, 1:500, BioGenes; anti-β-actin, 1:4000, Sigma). Alkaline phosphatase-conjugated anti-rabbit (1:4000, cell signaling) or anti-mouse antibody (1:3000, NEB) served as secondary antibodies.
Peptide-antibody production
A specific antibody was generated against the amino acids 18-31 of Robo3B (QFPCLNALRHPLSP) in rabbits by BioGenes GmbH (Berlin).
Cloning of Robo3A and Robo3B promoter region
Human Robo3 from −500 to −1 bp relative to the translation start was amplified by PCR (Robo3A: 5′-GACGGTACCGGGCAGAAGG-3′ and 5′-GACAAGCTTCTGCAGCAGCGTTTTC-3′; Robo3B: 5′- CCCTTGAAATGAAGCGTGATTATCC-3′ and 5′-CTCCTATGCTTCTCTGCGGAGC -3′) using Taq®-Polymerase (Roche, Mannheim, Germany). Subsequently, the amplified fragments were cloned into a pGL4basic vector (Promega Corporation, Madison, WI, USA) via HindIII-HF (NEB, Frankfurt am Main, Germany) and KpnI-HF (Robo3A) respectively EcoRV-HF (Robo3B) restriction sites. The vector sequences were confirmed by Sanger sequencing.
Reporter gene assays
The tumor cell lines (150,000–200,000 cells) were cultured in six-well plates for 24 h and then treated with cationic lipid/plasmid DNA suspension: 1 µg of luciferase reporter plasmid (Robo3A, Robo3B or an empty pGL4basic vector) and 0.1 µg of the internal control plasmid pRL-TK. Twenty-four hours after the transfection the cells were harvested and the lysate was analyzed for luciferase activity with a luminometer using Promega dual-luciferase assay reagent [14]. At least three independent transfection experiments were performed for each construct.
Cell treatments
The melanoma cell line Mel Ei (150,000 cells) were cultured in six-well plates and were treated for 24 h with different inhibitors (SB431542 InvivoGen 2 μM; S3I-201 Sigma-Aldrich 20 mM; Dorsomorphin Tebi-bio 2 mM; LY-294002 Sigma-Aldrich 20 mM; Wortmannin Sigma-Aldrich 10 mM; UO126 Calbiochem 10 mM; Vemurafenib Active Biochem 100 μM; KT5720 Merck Millipore 100 nM; Bisindolylmaleimide II Santa Cruz 10 μM; Gö 6983 Santa Cruz 10 μM; Wnt Agonist Calbiochem 10 μM). For the stability assay the cells were treated with α-Amanitin (AppliChem, 5 mM). After isolation of total RNA, quantitative RT-PCR was performed to detect relative gene expression. Normalization was based on mRNA input in this experiment.
Statistical analysis
Calculations were performed using the GraphPad Prism software (GraphPad software, Inc., San Diego, USA). All results are indicated as mean ± SEM and comparison between groups were made using the Student paired t test (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant).