U ch1

Human chordoma U-CH1, MUG-Chor1 and JHC7 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). U-CH1 and MUG-Chor1 cells were cultured in RPMI-1640 medium (Catalog No. 11875093, Gibco, US) supplemented with an additional 1% l-glutamine (Catalog No. 25030081, Gibco, US), 10% characterized fetal bovine serum (FBS) (Catalog No. 10099141, Gibco, US), 10 units/mL penicillin and 10 mg/mL streptomycin (Catalog No. 10378016, Gibco, US). JHC7 cells were cultured in DMEM: F12 medium (ATCC^® 30-2006™, ATCC, USA) supplemented with 10% FBS, 10 units/mL penicillin and 10 mg/mL streptomycin. To culture U-CH1 and MUG-Chor1 cells, coating buffer (50 μg/mL rat tail type I collagen (Catalog No. 354236, BD Biosciences) was added to the culture flask for 1 h at room temperature prior to adding the cells. The SGC-7901 (a gastric cancer cell line)-shN and sh393 cells and GES-1 (an immortalized gastric epithelial cell line)-shN and sh393 cells were obtained as previously described [32 (link)]. All cells were maintained in humidified incubators at 37 °C with 5% CO₂.

Human chordoma cell lines JHC7 and U-CH1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). JHC7 cells were cultured in DMEM: F12 medium (ATCC^® 30-2006™, ATCC, USA) and U-CH1 cells were cultured in RPMI-1640 medium (11875093, Gibco, US) supplemented with an additional 1% L-glutamine (25030081, Gibco, US). To culture U-CH1, coating buffer (50 μg/mL rat tail type I collagen (354236, BD Biosciences) was added to the culture flask for one hour at room temperature prior to adding cells. All cells were cultured with 10% characterized fetal bovine serum (FBS) (10099141, Gibco, US), 10 units/mL penicillin and 10 mg/mL streptomycin (10378016, Gibco, US), and maintained in humidified incubators at 37°C with 5% CO₂.
To knock down endogenous AURA, CDK9, and MOK, siRNA constructs were generated with the target sequences shown in Table S4. The siRNAs were purchased commercially (HanBio Inc., Shanghai, China). Chordoma cells were transfected with siRNA at a final concentration of 50 nM using Lipofectamine 3000 transfection reagent (Catalog No. L3000015, Invitrogen, USA). The suppression efficiency of AURA, CDK9, and MOK was analyzed by western blot on the third day post transfection.