Fig. 4: Knockdown of CALCB or RAMP1 inhibits proliferation of Ewing sarcoma (EwS) cells in vitro.

a Analysis of cell growth and cell death of A673 and RDES EwS cells with/without siRNA- or shRNA-mediated knockdown of CALCB. Left panel A673 and RDES: Given is the mean of relative cell count compared to Co (Control), which either received according doses of a non-targeting siControl or did not receive dox in assays with dox-inducible shRNAs (n = 3–5). SEM and unpaired two-tailed Student’s t test of relative total cell count. Right panel A673 and RDES: Knockdown of CALCB was verified by qRT-PCR. Given is the mean gene expression and SEM; unpaired two-tailed Student’s t test. b Colony-forming assays of A673 and RDES EwS cells with/without siRNA- or dox-induced shRNA-mediated knockdown of CALCB or RAMP1. Mean colony number and SEM normalized to control, which either received according doses of a non-targeting siControl or did not receive dox in assays with dox-inducible shRNAs (n = 3–6). Unpaired two-tailed Student’s t test. Representative images of each condition are shown. Knockdown of CALCB and RAMP1 were verified by qRT-PCR and are displayed in Fig. 4a and Supplementary Fig. S6. c Sphere-formation assays of A673 and RDES EwS cells with/without dox-induced shRNA-mediated knockdown of CALCB and RAMP1. Sphere index was calculated by addition of diameter of all existing spheres in one well divided by diameter of spheres in the control well. Mean and SEM (n = 3–6); unpaired two-tailed Student’s t test. n.s. P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001