Figure 1. HOXD13 expression in EwS is dependent on EWS::FLI1.

A) HOXD13 expression across different pediatric cancers (33, 34). B) qRT-PCR of Posterior HOXD genes in Ewing sarcoma cell lines and U2OS (osteosarcoma) cells. C) qRT-PCR and D) western blot of EWS::FLI1 96 hrs after knockdown. E) qRT-PCR of HOXD13, HOXD11, and HOXD10 expression in control and EWS::FLI1 knockdown cells. F) Fluorescent Immunocytochemistry of HOXD13 (Alexa647) in EWS::FLI1 knockdown cells. Nuclear counterstain was performed with DAPI. Scale bar is 50 um. G) Single-cell gene expression profiles from A673/TR/shEF xenografts (35) quantified by violin plots (Wilcoxon rank sum test). Error bars for qRT-PCR studies are representative of SEM from three independent replicates. Expression levels were determined relative to two housekeeping genes and fold changes expressed relative to control condition. * p<0.05; ** p<0.01; Two-way ANOVA; Sidak’s multiple comparison test; Two-tailed t-test. Atrt: atypical teratoid rhabdoid tumor, epd_it:Ependymoma infratentorial, etmr:embryonal tumor with multilayered rosettes, ews: Ewing sarcoma, ews-like: ewing-like, hgg_k27m: high-grade glioma K27M, hgg_other: high-grade glioma K27WT, mb_group3: medulloblastoma group 3, mb_group4: medulloblastoma group 4, mb_shh: medulloblastoma sonic hedgehog, mb_wnt: medulloblastoma wnt, nb: neuroblastoma, os: osteosarcoma, pa: pilocytic astrocytoma, rms: rhabdomyosarcoma, t-all:T-cell acute lymphoblastic leukemia