Crl 2814

L-Wnt5a CM was obtained from spent cultured medium of mouse fibroblasts L-M (TK-) cells transfected with a Wnt5a vector (CRL-2814, ATCC, Atlanta, United States). Mouse L-Wnt5A cells (∼2 × 10⁶ cells) were cultured in a T-75 flask containing GM until they reached 80% confluence. The following day, serum-free medium was replaced, cultured for 24 h and collected after sterile filtration as L-Wnt5A CM. Control CM was obtained from normal mouse fibroblast L-M (TK-) cells. All CMs were stored at −80°C. The ratio of 1:10 diluted WNT5A CM was used. Recombinant human WNT5A (0.01 μg/ml, R&D Systems) was supplemented to the GM of eMSC.

The Kuramochi cell line (RRID:CVCL_1345) was purchased from JCRB Cell Bank and cultured in RPMI 1640 medium (SH30027, HyClone) supplemented with 10% FBS, 50 units/ml penicillin, and 50 units/ml streptomycin (15140122, Gibco). Ascitic spheroids were cultured in MCDB/DMEM (1:1) medium (10372019 and 31966047, Gibco) supplemented with 10% FBS, 50 units/ml penicillin, and 50 units/ml streptomycin on ultra-low attachment plates (Corning). Cells and spheroids were stimulated with recombinant WNT proteins (645-WN-010, 5036-WN-CF; RnD Systems) at a concentration of 200 ng/ml or by WNT conditioned medium (CM) (control, WNT3A or WNT5A), produced by rat L-fibroblasts (CRL-2648, CRL-2647, CRL-2814; ATCC), at a ratio of 1:4 with complete culture medium. The Porcupine inhibitor LGK974 (974-02; Stem RD) was used at a 0.1 μM concentration while CK1 inhibitors PF670462 [sc-204180A; Santa Cruz Biotechnology (SCBT)] and D4476 (218696; Calbiochem) were used in 5 μM final concentration. If not stated otherwise, cells were pretreated with either LGK974 (+), or DMSO (-) for 24-48 h and treated with recombinant human (rh) WNTs/WNT CM overnight before being used for assays or WB.