SKN, which is a human uLMS cell line, and MES-SA, which is human uterine sarcoma cell line, were used in this study. SKN was purchased from Health Science Research Resources Bank (HSRRB, Osaka, Japan), and MES-SA was purchased from American type culture collection (ATCC, Virginia, USA). SKN cells were cultured in Ham's F 12 (Sigma-Aldrich Japan K.K., Tokyo, Japan), and MES-SA cells were cultured in McCoy's 5a medium (ATCC). Both media were supplemented with 10% heat-incubated fetal bovine serum (FBS). The cells were seeded at a density of 5×104 cells/well in a six-well plate, and incubated at 37°C in a humidified 5% CO2 incubator for 5 days. The cells were trypsinized and counted by a cell counter (Vi-CELL XR; Beckman Coulter, Tokyo, Japan) at each time point, as reported previously (36 (link)).
For TGF-β treatment, cells were cultured in Ham's F12 and McCoy's 5a medium supplemented with 10% FBS containing 100 or 500 pg/ml TGF-β1 (Sigma-Aldrich Japan K.K.) for 24 h. In order to block TGF-β signaling, SB431542 (WAKO, Tokyo, Japan), which is a TGF-β type I receptor-selective blocker, was dissolved at a concentration of 10 µM in dimethylsulfoxide (DMSO). Cells were seeded at a density of 5×104 cells/well in a six-well plate and cultured for 24 h and then cultured with new medium supplemented with 10 µM SB431542 for more 48 h.
For TGF-β treatment, cells were cultured in Ham's F12 and McCoy's 5a medium supplemented with 10% FBS containing 100 or 500 pg/ml TGF-β1 (Sigma-Aldrich Japan K.K.) for 24 h. In order to block TGF-β signaling, SB431542 (WAKO, Tokyo, Japan), which is a TGF-β type I receptor-selective blocker, was dissolved at a concentration of 10 µM in dimethylsulfoxide (DMSO). Cells were seeded at a density of 5×104 cells/well in a six-well plate and cultured for 24 h and then cultured with new medium supplemented with 10 µM SB431542 for more 48 h.