Ms023

HeLa and MCF7 cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM, WelGENE Inc.) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. All cell lines tested negative for mycoplasma contamination by PCR. siRNAs were transfected into HeLa cells using DharmaFect 1 (Dharmacon, Inc.). DNA transfection was performed using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. For inhibition experiments, the cells were incubated with the Haspin kinase inhibitor CHR-6494 (10 nM) (Calbiochem), the Aurora B inhibitor hesperadin (2 μM) (Selleckchem), the Cdk1 inhibitor RO3306 (1 μM) (Enzo Life Sciences), the proteasome inhibitor MG132 (0.5 μM) (Sigma-Aldrich), and the PRMT6 inhibitor MS023 (20 μM) (Sigma-Aldrich).

Purified MuSCs were seeded onto collagen-coated plates and expanded in growth media (Ham’s F10 media with 20% FBS, 2.5 ng/mL human recombinant bFGF, and 1% Penicillin/Streptomycin) at 37 °C and 5% CO₂. Media was replenished every two days. To differentiate myoblasts into myotubes, myoblasts were grown to 90% confluency in growth media, washed twice with 1 X PBS, and switched to differentiation media (DMEM, 1% FBS, and 1% Penicillin/Streptomycin). For inhibitor treatment, cells were incubated with 0.033% DMSO or 1 μM MS023 (Sigma) in 0.033% DMSO and media was replenished every second day for the duration of the indicated treatment time.