Fig. 7: AT3:VCP is a targetable functional dependency in cancer cells in vitro.

a Flow-cytometry for CFSE dye depletion by proliferation after siSCR, siTFE3, siVCP and combinations in FU-UR-1 cells. (siRNA#1 for each is represented by a solid line; siRNA#2 for each is represented by a dotted line.) b Clonogenic assays after siRNAs applied to both the FU-UR-1 cell line and the ASPS-1 cell line (black diamonds represent an siTFE3 that targets a portion of TFE3 not included in AT3). c CFSE dye depletion assay combining siRNA depletion of AT3 by siTFE3#1 (solid line) and siTFE3#2 (dotted line) or control siSCR with CB-5083 or vehicle, showing that AT3 depletion or CB-5083 diminishes proliferation (AT3 depletion slightly more than CB-5083), but that their combination further diminishes proliferation (pushes distribution further to the right). (siRNA#1 for each is represented by a solid line; siRNA#2 for each is represented by a dotted line.) d The same for siVCP#1 (solid line) and siVCP#2 (dotted line) or control siSCR, with CB-5083 or vehicle, showing the same phenomenon. e Schematic of the Rosa26-LSL-AT3-IRES-eGFP allele that is activated by Cre-recombinase-mediated excision of a stop sequence between the AT3 coding sequence and the native Rosa26 promoter. This allele, when activated in living mice led to the fully penetrant development of ASPS tumors. f Flow-cytometry for CFSE dye depletion by proliferation in GFP+ sorted mouse embryonic fibroblasts (MEFs) that bear an ASPSCR1-TFE3 allele that is induced by Cre-mediated recombination to remove a stop sequence between the promoter and the cDNA coding sequence. (Cells counted at day 0 were 9,073; ells counted at day 4 were 20,258 for TATCre added alone, 20,241 for TATCre and VCP-GFP added, 19,982 for VCP-GFP added alone, and 14,346 for GFP control added alone).