Pathogenic mutations in ATP8B1 cause cell-autonomous defects in intestinal lumen formation. (A) Maximum intensity projections of WT and PFIC small intestinal organoids fixed and stained with phalloidin (green) and DAPI (blue). (B) Ratio of apical versus basal membrane length of WT (n = 11) and PFIC organoids (n = 15). *, P < 0.0002. (C) Western blot of lysates from WT and PFIC organoids probed for ATP8B1 and Na/K ATPase. (D) PFIC organoid staining for the enterocyte marker villin, phalloidin, and DAPI. (E) WT and PFIC organoids stained for the apical endosomal marker myosin Vb. (F) Control and PFIC1 patient ileum samples stained for myosin Vb. Red arrows highlight apical myosin Vb staining. (G) Ileal sample from PFIC1 patient immunostained for myosin Vb. Red arrows highlight villi, and green arrows highlight crypts. Bars: (A, D, and E [left]) 35 µm; (E, zoom) 10 µm; (F and G) 25 µm.