Table 1.
Primer Sequences for Real Time PCR.
Figure 1.
Three-dimensional Caco-2 spheroids express the TJ, the AJ proteins and the peri-junctional actin ring.
[A] Protein expression of ZO-1 and occludin was detected using immunofluorescence microscopy (original magnification, ×63) with mouse anti- ZO-1 (green), rabbit anti-occludin (red) followed by DAPI nuclear stain (blue). [B] Protein expression of E-cadherin and ß-catenin was detected using immunofluorescence microscopy (original magnification, ×63) with mouse anti-E-cadherin (green), rabbit anti-ß-catenin (red); and nuclei were counterstained with DAPI. [C] Actin filaments were detected using fluorescence microscopy (original magnification, ×63) with phalloidin (red) and nuclei were counterstained with DAPI (blue). Representative images captured from cross-section of the spheroids are shown. The bars indicate 10 µm.
Figure 2.
Transmission electron microscopy analysis of Caco-2 cells spheroids.
A junctional complex (tight junction; TJ, adherens junction; AJ, D; desmosome) between adjacent cells is evident, apically [A]. The 3-D culture resulted in a luminal space (L) with formation of microvilli (mv) at the apical side and polarization of cells located in the cortical region of spheroid is indicated by supranuclear localization of Golgi (G) apparatuses [B].
Figure 3.
Ethanol and acetaldehyde and in combination increase permeation of the fluorescent marker FD4 from the basolateral to the luminal side of Caco-2 spheroids.
[A–C] Spheroids were exposed at the basal side, in the presence of FD4, to medium only (−ve control), 2 mM EGTA (+ve control) and either 10 mM, 20 mM, 40 mM ethanol (panel A), 25 µM, 50 µM, 100 µM or 200 µM acetaldehyde (panel B) or in combination (panel C). Intraluminal accumulation of FD4 (green) was measured using confocal microscopy (original magnification, ×63) and representative images captured from the middle transsection of spheroids are shown and the bars indicate 10 µm. Ethanol, acetaldehyde and in combination increase FD4 permeation dose-dependently in Caco-2 spheroids. [D–F] The mean fluorescence intensity of FD4 from 8 spheroids was measured and expressed as the ratio of the luminal (L) over the basal (BL) compartment. The L/BL ratio of EGTA exposure (maximal TJ disruption) was set to 1. All graphs indicate the results of three replicate experiments. Data were expressed as means±SD, for ethanol (D), for acetaldehyde (E) and for combination (F), *P<.0001.
Figure 4.
Ethanol and acetaldehyde exposure alters ZO-1 and occludin distribution at tight junctions of the Caco-2 spheroids.
[A] Spheroids were exposed to medium only as control, 40 mM ethanol or 200 µM acetaldehyde for 3 h and labeled for ZO-1 (green), occludin (red) and nuclei (blue) by confocal immunofluorescence staining (original magnification, ×63) and representative images captured from the middle transsection of spheroids are shown. Bars indicate 10 µm. Effects of ethanol and acetaldehyde on TJ morphology. [B] Caco-2 spheroids were exposed to medium only, either ethanol or acetaldehyde for 3 h, fixed and processed for Transmission electron microscopy. The lateral surface is indicated by arrows. Bars indicate 0.5 µm.
Figure 5.
Hyperacetylation of α-tubulin and lysine residues, and localization of ZO-1 in Caco-2 spheroids.
Protein expression of acetylated-α-tubulin, acetylated-lysine residues and ZO-1 detected using immunofluorescence microscopy (original magnification, ×63) with mouse anti-α-tubulin (green), rabbit anti-acetylated-lysine (bred), and nuclei were counterstained with DAPI (blue) and mouse anti-ZO-1 (green). Representative images captured from cross-sections of the spheroids are shown. The bars indicate 10 µm.
Figure 6.
Ethanol and acetaldehyde did not alter expression of the tight junction-coding genes in Caco-2 spheroids.
Spheroids were incubated with medium only (control), 40 mM ethanol and 200 µM acetaldehyde. Data were expressed as means of three replicates ± SD; all P values>0.05 comparing ethanol or acetaldehyde vs. control.