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

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

<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