Noggin

Cells were scraped into lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 50 mM Tris–HCl (pH 6.8). Lysates were quantitated and equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were then transferred onto Polyvinylidene fluoride (PVDF) membrane and immunoblotted with specific antibodies. Antibodies used were PPARγ, phosphor-p38 MAPK, p38 MAPK kinase, phosphor-Smad1/5/8, Smad1, Smad4, Pref1, phosphor-Akt, Akt, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-p65, p65 (Cell Signaling Technology), BMPRIA (Abcam), Noggin (Proteintech), B4GalT5 (Sigma), Hsp90 (Santa Cruz Biotechnology), and C/EBPα, and 422/aP2 (obtained from the Department of Biological Chemistry at the Johns Hopkins University School of Medicine).

The organoid culture was performed in accordance to the protocol described previously [21 ]. In brief, organoids were generated from isolated crypts of the colon of colitis mice (C57/BL6 mice and Lrrc19^−/− mice) and then embedded into Matrigel (Corning, Corning, New York, USA). After that, organoids were kept in Organoid Growth Medium (STEMCELL Technologies) in the presence of R-Spondin, Noggin, and EGF (Proteintech). To investigate the effect of DVF on organoids, organoids were co-cultured with 1 μg DVF or PBS on 6-well plates. After 5 days of co-culture, organoid morphologies were recorded and then harvested for further experiments.