(A) Hierarchical clustering of CD99-associated signature based on TC-71 and IOR/BRZ silenced cells. The signature included 106 genes: 76 upregulated and 30 downregulated (Supplemental Table 1; t test, P < 0.05). The top 10 CD99 up- (red) or downmodulated genes (blue) are shown. (B) We applied PCA using the CD99-associated signature (Supplemental Table 1) on EWS/FLI1-silenced EWS cells derived from SK-M-NC and EW24 (dataset from ref. 10) or from EWS502 and TC-71 cell lines (dataset from ref. 55). CD99 signature allowed for a clear separation of EWS/FLI knockdown cells from controls. Circles indicate EWS/FLI1 silenced cells; triangles indicate controls. (C) Interaction between EWS/FLI1 and CD99 promoter (prom). ChIP was carried out in TC71 and IOR/BRZ cells as described in Supplemental Methods by using anti-Fli1 antibody. The CD99 promoter region containing the ets sequence was detected by PCR with specific primers listed in Supplemental Methods. One microliter of initial preparations of soluble chromatin (Input) was amplified to control input DNA. In control samples (N), normal rabbit IgG was used instead of the primary Ab as control of Ab specificity. The occupancy of EWS/FLI1 on the TGFβR2 promoter was tested with specific primers as a control for ChIP reaction. The negative control promoter primers used for ChIP were previously described (72).