Fig 2: Piperacetazine inhibits endogenous PAX3::FOXO1 activity in multiple cell lines. A, The effect of piperacetazine treatment on cell viability was measured using the CellTiter-Blue assay (black lines). The effect of piperacetazine treatment on PAX3::FOXO1 activity was measured via luciferase assay (blue lines), using the PAX3::FOXO1-responsive pGL3-PDGFRA luciferase reporter (RH30, RH41, RH28, U66788, RD), and PGK reporter (SN12C, negative control). B, Left: Luciferase activity was measured in HEK293T cells transfected with PAX3::FOXO1-responsive ASS1P reporter and an empty or PAX3::FOXO1 expression vector. Cells were treated with DMSO or piperacetazine for 48 hours. Matching Western blots are shown under each bar. Right: The same experimental setup was repeated with EWS::FLI1-responsive NR0B1 luciferase reporter and an EWS::FLI1 expression vector or empty vector. The cells were treated with the same concentration of piperacetazine or DMSO for 48 hours, and the normalized luciferase readouts and accompanying Western blots are shown. (*, P < 0.0001; Student t test; ns, P > 0.05)