Fig. 1: AT3 interacts with VCP/p97 in the nucleus of ASPS and RCC tumor cells through its ASPSCR1 portion.

a Plot of enrichment scores (defined in Methods) for homologous proteins identified on mass spectroscopy proteomics following ASPSCR1 immunoprecipitation (IP) of nuclear lysates from human FU-UR-1 cells (n = 8 + 2 controls IgG) and mouse ASPS tumors (n = 7 + 3 controls IgG) b IPs followed by western blots (WB) showing reciprocal AT3 (by ASPSCR1 or TFE3 antibodies) and VCP interactions in multiple tumor cell circumstances, but not control HCT116 cells (input = 10% of sample; FT = flow-through; IP-IgG and FT-IgG lanes are mock IPs with non-specific IgG; each IP-WB was repeated on a biologically independent sample; all uncropped gel images provided in Source Data file in Supplemental Information). c IP-WBs demonstrating reciprocal interactions in multiple tumor contexts between AT3 and MATR3 or MTA2 (n = 2 biological repeat not shown). d ASPSCR1, TFE3, and AT3 constructs oriented left-to-right:amino-to-carboxy. Gray background coded by ASPSCR1; slashes indicate amino acids 1-311. White background coded by TFE3; slashes indicate exon 4 through 3’-terminus; black coded by exon 3, the putative activation ___domain. e FLAG-IP-WB for MATR3, MTA2, and VCP in HEK cells transfected with FLAG-tagged constructs. Expression varied on n = 3 biological repeats: more AT3.2 was expressed in the iteration of the experiment depicted. f Fluorescence photomicrographs of a human ASPS tumor and control clear cell sarcoma (CCS) and Ewing sarcoma (ES) tumors stained with DAPI, TFE3 (detecting AT3), and VCP antibodies. (panel = 100 µm square; n > 4 fields per each of n = 4 tumors/controls). g Fluorescence images of HEK293T co-transfected with VCP-GFP and either ASPSCR1-TFE3-mRFP or mRFP alone. (panel = 30 µm square). Nuclear (nuc.) and cytoplasmic (cyt.) fraction western blots (WBs) for VCP (n > 4 fields per n = 2 biological repeated experiment). h Fluorescence photomicrographs of proximity ligation assay (PLA) between TFE3 and VCP antibodies in the ASPS tumor and controls, with an additional IgG-PLA control. (panel=100 µm square; n > 4 fields per each of n = 4 tumors/controls). i Quantitative fluorescent PLA signal per nucleus was measured in (79, 57, 143, 29, 105, 81, 83, 57, 216, 271, 215, 244, 84, 59, 195, 65 nuclei for the samples listed in order.).