Skut 1

Primary patient-derived LMS cell lines (STS39, STS54, STS137, STS210, STS551) and UPS cell lines (STS148, STS162, STS235, STS309, STS548) were established from pathologically reviewed surgical specimens in accordance with institutional research ethics as described elsewhere⁶⁰ . SKLMS-1, SKUT1, and SKUT-1B and Hs 789.Sk cell lines were obtained from ATCC (Manassas, VA, USA). RPEΔp53 and RPEΔp53ΔBRCA1 were kind gift of D. Durocher. All cell lines are routinely authenticated by STR-analysis at The Center for Applied Genomics (TCAG), SickKids in Toronto, and tested negative for mycoplasma (ABM, Canada). All cases were reviewed by a dedicated sarcoma pathologist (BCD) (See Supplementary Data 8 and 9). LMS cell lines maintained smooth muscle expression (Supplementary Fig. 25). STS39, STS54, STS137, STS210, STS551, STS148, STS162, STS235, STS309, STS548, and SKLMS1 cells were cultured in GlutaMAX-supplemented DMEM/F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) +1% Penicillin/Streptomycin (Thermo Fisher Scientific) and 10% heat-inactivated fetal calf serum (FBS) (Wisent, St-Bruno, Canada) at 37 °C, 5% CO2. SKUT1, SKUT-1B, RPEΔp53 and RPEΔp53/ΔBRCA1 were cultured in GlutaMAX-supplemented DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) +1% Penicillin / Streptomycin and 10% FBS at 37 °C, 5% CO2.

Human uterine sarcoma cell lines SK-UT-1 and MES-SA were obtained from ATCC. MES-SA was cultured in Gibco™ DMEM (Carlsbad, CA), and SK-UT-1 was cultured in Gibco™ MEM supplemented with 10% FBS (Grand Island, NY, USA). SHARPIN siRNA (target sequence 5’-CCCTGAGTGTTCAGCTTCA-3’) and negative-control siRNA (target sequence 5’-GGCTCTAGAAAAGCCTATGC-3’) were transfected into cells (5 µg siRNA, ratio 1:1 duplexes) using ExFect2000 Transfection reagent (Vazyme, Nanjing, China) for 72 h according to the manufacturer’s instructions and were subsequently collected for Cell Counting Kit-8 (CCK-8), Western blot (WB) and colony formation assays.