Calcium mobilisation upon NMURs activation in CRC cells

Neuromedin U is a small secreted peptide recently associated with several cancer types. NMU receptors (NMUR1, NMUR2) belong to the superfamily of G protein-coupled receptors (GPCRs). They have high homology, and according to previous reports, they both induce the same signalling pathways involving inositol phosphates and calcium as secondary messengers. We tested calcium mobilisation upon NMU-9 or NMU analogues treatment of colorectal cancer cell lines with endogenous expression of NMU receptors.PBS rinsed cells were loaded with Fluo-4 AM (5 μM; Invitrogen) for 45 - 60 min at 37 °C in the presence of 0.5 % of pluronic-127 in the incubation medium (growth medium without FBS and PS, supplemented with 0.1 % BSA). Cells were rinsed with warm PBS and bathed in a final volume of 300 µl of incubation medium. Images were acquired using AxioVert fluorescence inverted microscope (Zeiss, Germany) with the use of 10 x dry objective (NA 0.3). A time-lapse image sequence in green channel (Fluo-4) was recorded with exposure time of 500 ms in the same field of view. After approximately 20 s of acquisition peptide was added (type of the peptide and concentration are included in the file title) and acquisition was performed. We used: NMU-9 (Phoenix Pharmaceuticals), the NMUR1 agonist SBL-NMU-21 and NMUR2 agonist SBL-NMU-17 (De Prins A., Eur J Med Chem. 2018).