The role of FeCl₃ on calcium transport across Caco-2 monolayer pre-treated with 1,25(OH)₂D₃ and ascorbic acid (Asc).

(A) Experimental timeline (please see text for detail). (B–E) Transepithelial calcium transport and epithelial electrical parameters (PD, Isc, and TER) in Caco-2 monolayers with or without 10 nM 1,25(OH)2D3, 200 μM FeCl3, and 0.5 mM Asc. PD values were the magnitudes of potential difference (the apical side being negative with respect to the basolateral side), and glucose made the apical side more negative. (n = 10; *P P ††P †††P 2D3-treated group (blue bar).</p

Expression of genes and proteins related to transepithelial calcium transport, and transepithelial resistance in Caco-2 cells.

(A) Expression of TRPV6, calbindin-D9k, PMCA1b and NCX1 mRNA, as determined by quantitative real-time PCR. Representative fluorescent photomicrographs from conventional fluorescent microscope (400×; Nikon model Eclipse Ni-U) and confocal laser scanning microscope (630×; Carl Zeiss model Zeiss LSM800) confirmed the presence of TRPV6 (red) and PMCA1b (green) protein expression in 3-day Caco-2 monolayer [blue, nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI)]. The drawing diagram shows localization of calcium transporters in a Caco-2 cell. TRPV6, transient receptor potential vanilloid subfamily member 6; PMCA1b, plasma membrane Ca2+-ATPase 1b; NCX1, Na+/Ca2+ exchanger 1; NKA, Na+/K+-ATPase. (B) Transepithelial resistance (TER) of Caco-2 cells after being cultured on Snapwells for 3 or 6 days (n = 4/group; two-tailed, unpaired Student’s t-test). NS, not significant.</p

The effects of VIP/pituitary adenylate cyclase-activating polypeptide receptor (VPAC) agonist on intestinal epithelium-like Caco-2 monolayers.

(A) Timeline of Experiment 1. (B) Transepithelial calcium flux and (C–E) epithelial electrical parameters (PD, Isc, and TER) across Caco-2 monolayers that were directly exposed to 0.1 and 1 μM VPAC agonist on the basolateral side for 30 min. *p < 0.05 vs. control group (open bar, one-way ANOVA with Tukey’s multiple comparison test).</p

<i>Homo sapiens</i> primers used in real-time PCR.

The sequences of primers of calcium transport-related genes. (DOCX)</p

<i>In silico</i> molecular docking and the effects of CFTR activator (genistein) on transepithelial calcium transport.

(A) The architecture of CFTR and location of the proposed CFTRinh-172 (magenta)- and genistein (green)-binding pockets. (B) CFTRinh-172 (magenta) surrounded by His199-Arg74-Trp79-Trp202-Trp361 (yellow) resembles the thiazolidinyl ring’s binding interaction from the PDB ID: 4JIR and PDB ID: 4JIH. (C) Genistein (green) was docked to the Arg297-Asp984/5-His949 motif (cyan) in the orientation found in PDB ID: 1X7J. (D) Transepithelial calcium flux and (E–G) epithelial electrical parameters (PD, Isc, and TER) across Caco-2 monolayers directly exposed to 50 μM genistein (Gen.) on the apical side for 30 min. *p t-test).</p

The effects of VIP/pituitary adenylate cyclase-activating polypeptide receptor (VPAC) inhibitor on intestinal epithelium-like Caco-2 monolayers.

(A) Timeline of Experiment 2. (B) Transepithelial calcium flux and (C–E) epithelial electrical parameters (PD, Isc, and TER) across Caco-2 monolayers pretreated with 10 nM 1,25(OH)2D3 for 72 h and then exposed to 1 and 10 μM VPAC inh. on the basolateral side for 30 min. *p p p †p 2D3-treated group (closed bar, one-way ANOVA with Tukey’s multiple comparison test).</p

The effects of an CFTR inhibitor on transepithelial calcium transport.

(A) Timeline of Experiment 3. (B) Transepithelial calcium flux and (C–E) epithelial electrical parameters (PD, Isc, and TER) across Caco-2 monolayers pretreated with 10 nM 1,25(OH)2D3 for 72 h and then exposed to 2–200 μM CFTR inhibitor (CFTRinh-172) on the apical side. ***p †p †††p 2D3-treated group (closed bar, one-way ANOVA with Tukey’s multiple comparison test).</p

Proposed CFTR_inh-172- and genistein-binding pockets of CFTR channel.

A video showing the architecture of a CFTR channel and the sites of proposed CFTRinh-172- and genistein-binding pockets (magenta and green, respectively). The movie was created by using PyMOL 1.7.4 (The PyMOL Molecular Graphics System, Schrödinger, LLC.). (ZIP)</p