Supplementary Figure 4: Knockdown of EGR2 reduces clonogenic growth, cell cycle progression, and viability of Ewing sarcoma cells.

(a) Analysis of EGR2 protein expression by immunoblot in Ewing sarcoma cell lines (loading control: β-actin). The neuroblastoma cell line SK-N-SH served as negative control. (b) Analysis of EGR2 protein expression by immunohistochemistry in tumors of xenografted Ewing sarcoma cell lines (A673, TC-71 and SK-ES1), the alveolar rhabdomyosarcoma cell line SJ-RH30, and the neuroblastoma cell line IMR-32, and comparison with EGR2 mRNA expression levels as determined by Affymetrix HG-U133Plus2.0 arrays (GSE36133). Scale bars = 200 µm. (c) qRT-PCR analysis of knockdown efficacy of siRNAs used to silence EGR2 or ADO (48 h after transfection). Data are shown as the mean and s.e.m.; n ≥ 4 independent experiments. (d) Analysis of clonogenic growth after seeding at low-density and serial re-transfection with siRNA every four days. Cells were fixed 9–14 days after seeding and individual colonies were colorized with crystal violet. (e) Analysis of cell cycle phases by PI staining 96 h after transfection with siRNA. EGR2 knockdown reduces the percentage of cells in S phase (P < 0.01 for A673, SK-N-MC, and POE; P = 0.12 for EW7), while increasing the percentage in sub G1 phase (all P < 0.0001). (f) Analysis of apoptosis by Annexin-V-staining 96 h after transfection with siRNA. Data in d-f are shown as the mean and s.e.m. of results obtained with two different siRNAs for EGR2 and three different siRNAs for ADO as displayed in c; n ≥ 3 independent experiments. Two-tailed unpaired Student’s t-test; ns, not significant; *** P < 0.001.