Matrigel

The Matrigel plug assay was performed as described previously [29 (link)]. To further evaluate the angiogenic effect of THZ1 in vivo, liquid Matrigel (Corning Life Science) containing heparin (20 U) and VEGF (100 ng) with or without THZ1 (250 nM) were injected into FVB (Friend leukemia virus B) mice (n = 3, obtained from the Taiwan National Laboratory Animal Center). Matrigel with PBS plus heparin served as negative controls, whereas VEGF plus heparin was used as positive controls. Five days after administering the injection, the mice were sacrificed by CO₂ asphyxiation. The Matrigel plugs were excised and photographed. For further analysis of angiogenesis, hemoglobin content in Matrigel plug was measured with hemoglobin colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI, USA). Briefly, Matrigel plugs were weighted and then homogenized in 1 mL deionized H₂O for 5–10 min on ice. After centrifugation, supernatants were collected and mixed with hemoglobin detector at room temperature for 15 min. After incubation, the concentration of hemoglobin in Matrigel plugs was calculated with a standard curve by measuring the absorbance at 560 nm according to manufacturer’s protocol. Furthermore, the calculated values were normalized with plug weight and expressed as g/dl hemoglobin per milligram Matrigel plug.

Organoids were cultured as described previously with slight modifications^{37 (link)}. Sorted EpCAM⁺ cells were mixed with Matrigel (Corning, Corning, NY) containing 750 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ), 1.5 μg/mL Noggin (Miltenyi Biotec, Germany), and 15 μmol/L Jagged-1 (Anaspec, Fremont, CA). The cells were seeded on 96-well plates at 3,000 cells/10 μL of Matrigel per well. After the Matrigel polymerized, 100 μL of culture medium consisting of Advanced Dulbecco’s Modified Eagle Medium/F12 supplemented with 100 U/mL penicillin/streptomycin, GlutaMAX, 10 mmol/L HEPES, 1 × N2, 1 × B27, 1 mmol/L N-acetylcysteine, 2.5 μmol/L Thiazovivin (Cayman, Ann Arbor, MI), and 2.5 μmol/L CHIR99021 (Axon Medchem, The Netherlands) was added to each well. On day 2 of culture, 50 μL of culture medium containing 10% (v/v) R-spondin 1 culture supernatant and growth factors (1 μM Jagged-1, 50 ng/mL epidermal growth factor, and 100 ng/mL Noggin at final concentration) was added to each well. On day 6, the number of organoids was counted and photographed in each well. The R-spondin 1 culture supernatant was harvested from the 293T-HA-RspoI-Fc cell line, which was provided by Calvin Kuo of Stanford University.