Porcupine inhibitor iwp 2

Cells were plated into 96-well white-walled plates (Greiner Bio-One) at 1×10³ cells/well and allowed to adhere for 24 hours before drug treatments. The cells were then exposed to serial dilutions of doxorubicin (0–10 µM, LC Labs) and were placed in either normoxic or hypoxic conditions (as described above). After an additional 72 hours, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability assay (Promega). Data were normalized to an untreated control well and graphed, and half maximal inhibitory concentration (IC₅₀) values were calculated from the dose-response curve as the concentration of doxorubicin that produced a 50% decrease in the mean luminescence relative to untreated control wells. Statistical tests (two-tailed paired t-test and two-tailed two-sample t-test) were performed using R with significance set at α = 0.05. The tankyrase inhibitor XAV939 (Santa Cruz) and porcupine inhibitor IWP-2 (Sigma Aldrich) were used at a 10 µM concentration, with dimethyl sulfoxide (DMSO; Fischer) alone used as a control.

Three-dimensional organotypic culture was performed in growth factor-reduced BM from Engelbreth–Holm–Swarm mouse sarcoma (Matrigel, Becton Dickinson), which was prepared according to the manufacturer's instructions. Isolated primary MMECs grown on low adhesion plates were trypsinized with 0.05% Trypsin-EDTA for 5-10 min, centrifuged and suspended with liquid Matrigel, and plated onto 8-well chamber slides at ∼1500 cells/well. Organoids were grown in DMEM/F12 supplemented with ITS medium supplement (Sigma-Aldrich), penicillin and streptomycin and 2.5 nM FGF-2 (Sigma-Aldrich). Wnt3a and Wnt4 (both R&D Biosystems) were used in 100 ng/ml, Rspo1 (R&D Biosystems) in 500 ng/ml, and porcupine inhibitor IWP-2 (Sigma-Aldrich) in 2 µM, and were added on the starting day of the cultures. Medium was refreshed every 3-4 days. For lactogenic differentiation, organoids were first grown for 6 days in the DMEM/F12 medium supplemented with 2.5 nM FGF-2, ITS and penicillin and streptomycin, and thereafter, 6 days in the same culture medium additionally supplemented with 1 μg/ml mouse prolactin (Sigma-Aldrich) and 1 μg/ml hydrocortisone (Sigma-Aldrich).