Experimental schema for lineage-specific transcription factor silencing and expression. (A) Ewing Sarcoma (EWS502) cells and primary human endothelial cells (HUVEC) were transduced with lentivirus expressing FLI1 3′ UTR-directed shRNA and HA epitope-tagged versions of EWS–FLI or FLI1. (B) Anti-FLI1 or anti-HA immunoblots of Ewing Sarcoma cells (EWS502) or endothelial cells (HUVEC) demonstrating concurrent silencing and replacement with HA–EWS–FLI (EF) or HA–FLI1. Tubulin serves as a loading control. Asterisks indicate where a background band runs at a similar molecular weight as endogenous FLI1. (C) After EWS–FLI1 silencing alone (Knockdown) or together with ectopic EWS–FLI1 or FLI1 expression, EWS502 cells were counted. EWS–FLI expression, but not FLI1, rescues the effect of knockdown on proliferation.