Fig. 6: Blockage of the calcitonin gene-related peptide (CGRP) receptor by small molecule inhibitors mimics the effect of CALCB and RAMP1 knockdown in vitro.

a Analysis of cell viability (normalized Resazurin signal to DMSO control) in A673 Ewing sarcoma (EwS) cells treated for 72 h with the indicated concentrations of MK-3207. The graph shows the dose-dependent relative Resazurin signal. Data are represented as mean and SEM (n = 12); unpaired two-tailed Student’s t test. b Comparison of relative Resazurin signal of A673 cells carrying a dox-inducible shRNA against RAMP1 treated with 150 µM of MK-3207 with/without knockdown of RAMP1 by additional addition of 1 µg/ml dox to the growth medium. Data are represented as mean and SEM (n = 3); unpaired two-tailed Student’s t test. c Analysis of colony-forming capacity of A673 (left panel) and RDES (right panel) EwS cells under treatment with the small molecule CGRP receptor inhibitors MK-3207 (20 µM) or BIBN-4096 (Olcegepant; 100 µM). DMSO served as control for treatment. Representative images of the colonies are shown below. Data are represented as mean and SEM (n = 3); unpaired two-tailed Student’s t test. d Analysis of sphere-formation capacity of A673 (left panel) and RDES (right panel) EwS cells under treatment with the small molecule CGRP receptor inhibitors MK-3207 (20 µM) or BIBN-4096 (Olcegepant; 100 µM). DMSO served as control for treatment. Sphere index was calculated by addition of diameters of all existing spheres in one well divided by diameter of spheres in the control well. Data are represented as mean and SEM (n = 3); unpaired two-tailed Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001