Stable MDA/RLN2 transfectants secrete bioactive RLN2 and express functional RXFP1. (a) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed showing increased expression of RLN2 transcripts in all five MDA/RLN2 transfectants when compared with MDA/EGFP-vector controls. Employing intron spanning primers encoding for the relaxin receptor RXFP1, a single transcript encoding RXFP1 was detected in both MDA/EGFP and MDA/RLN transfectants. 18S transcript levels were used as reference. (b) Secreted relaxin was quantified in the culture medium using a human relaxin-ELISA. MDA/RLN2 transfectants secreted significantly higher amounts of RLN2 compared with the MDA/EGFP control; ** p ≤ 0.005. (c) A cAMPassay was used to determine the bioactivity of secreted RLN2. Intracellular cAMP concentration was determined in MDA-MB-231 cells following incubation with 100 ng/ml rhRLN, the culture supernatants of MDA/RLN2 clone 23 (SN-RLN-23) or of MDA/EGFP (SN-EGFP) transfectants. Forskolin (10 μM) was used as positive control and untreated cells served as negative control. Induction of cAMP by secreted RLN2 and rhRLN2 demonstrates the presence of a functional RXFP1 receptor system in this cell line. Experiments were performed in triplicates; data were presented as mean ± SEM. Statistical significance was determined with student t-test and p ≤ 0.05 were marked with an asterisk. (d) The presence of RXFP1 protein in stable MDA/EGFP and MDA/RLN2 transfectants was detected by immunocytochemistry using a rabbit polyclonal antibody to human RXFP1. Positive staining for RXFP1 was obtained for all cell clones and shown here for (ai) MDA/EGFP, (bi) MDA/RLN2-4 and (ci) MDA/RLN2-19. (di) A negative control using a rabbit non-immune serum did not result in any staining.