Fig. 2. BAs promote NPC1L1 transportation to PM.

a The topology of human NPC1L1-3×Myc-EGFP. The 3×Myc tag was inserted after the 966th amino acid of NPC1L1 and faced the extracellular/luminal side. A₁₂₇₂LAL, Q₁₂₇₇KR and Y₁₃₀₆VNxxF were cytosolic amino acids of NPC1L1. The EGFP protein was fused with the carboxyl terminus of NPC1L1. SSD: sterol-sensing domain. b CRL1601/NPC1L1-3×Myc-EGFP cells were treated with bile, dialyzed bile, boiled bile, BA mixture, GDCA or CDX for 2 h. The surface NPC1L1 was labeled by anti-Myc antibody prior to permeabilization, and cellular cholesterol was stained by filipin. Scale bar, 10 μm. Percentages of ERC cholesterol relative to total cholesterol (c) and relative intensity of surface NPC1L1 (d) in (b) were quantified. The ERC cholesterol was defined as the cholesterol colocalized with intracellular NPC1L1. The average intensity of surface NPC1L1 in GDCA-treated cells was defined as 1. Values were presented as mean ± SD (n = 3 independent trials, 100 cells/trial). One-way ANOVA with Tukey post hoc test, ***P < 0.001; ns, no significance. e Filipin staining of in vitro cultured intestine sections from Npc1l1-EGFP knock-in mice. Small intestine from neonatal mouse was incubated with or without 10 μg/mL cholesterol in the presence or absence of indicated BA species or BA mixture for 2 h, and then fixed by 4% PFA (n = 2). Chol, cholesterol. Boxed areas are shown at a higher magnification on the right. Scale bar, 10 μm (main); 1 μm (inset). Deparaffinized sections of intestinal samples from mice receiving sham surgery or bile duct cannulation were stained with anti-NPC1L1 and anti-Villin (f), or with anti-Rab11 antibodies (g), followed by counterstaining with Hoechst (n = 2). Boxed areas are shown at a higher magnification on the right. Scale bar, 10 μm (main); 1 μm (inset). h Filipin staining of intestinal sections from Npc1l1-EGFP knock-in mice receiving sham surgery or bile duct cannulation (n = 2). The control group received sham surgery. Chol, cholesterol. Boxed areas are shown at a higher magnification on the right. Scale bar, 10 μm (main); 1 μm (inset). Chol: Cholesterol. Source data are provided as a Source Data file.