Fig 2: Proteins more abundant upon cysteine cathepsin inhibition. The connectivity of all proteins more abundant (log2 Fc ≥ 0.58) in the quantitative proteome comparison of TGFβ-1 treated to TGFβ-1 + E64d treated iPL32 cells was determined with STRING (Search Tool for the Retrieval of interacting Genes/Proteins). The three clusters most significantly enriched: lysosome p = 2.3 x 10−12, endosome p = 4.6 x 10−7, proteasome p = 4.2 x 10-12, and Jam-a “f11r” are highlighted. Different line colors indicate the type of evidence for interaction.

Fig 3: JAM-A Barrier Promoting Signaling Pathway and Perturbations Induced by Ethanol. JAM-A associates directly with ZO-2, which forms a complex with Afadin and the guanine-nucleotide exchange factor PDZ-GEF1. This complex activates Rap2c by promoting GTP binding. Active Rap2c inhibits RhoA-mediated activation of MLCK and downstream MLC phosphorylation which promotes barrier function. Ethanol exposure disrupts upstream targets, disinhibiting RhoA, and resulting in enhanced MLC phosphorylation. Contraction of the apical actomyosin ring follows, leading to an expanded paracellular space and increased solute flux. Perturbations induced by ethanol depicted as red lightning bolts. Arrows indicate activation, whereas bar-headed lines indicate inhibition.

Fig 4: Ethanol induces a dose- and time-dependent decrease in JAM-A and occludin, but not ZO-2, protein expression in Caco-2 and SK-CO15 monolayers. Caco-2 monolayers 15-day after seeding were incubated overnight followed by treatment with 0% (medium), 3%, or 6% v/v ethanol for an additional 6-h incubation (A). Additional transwell plates were exposed to 6% ethanol in staggered fashion for 0- (medium alone), 3-, or 6-h (B). SK-CO15 monolayers were also treated with 0%, 3%, or 6% v/v ethanol for 6 h (C) Total protein lysates were collected and analyzed by western blot. Images are representative of results of at least 6 (A), 4 (B), and 3 (C) independent experiments. Densitometry results are reported as the fold-change in target protein expression (normalized to ß-actin) relative to monolayers treated with medium alone. Densitometry data are reported as means ± SEM and analyzed by one-way ANOVA, *P < 0.05, ***P < 0.001.

Fig 5: JAM-A overexpression in Caco-2 monolayers is associated with increased TEER and enhanced barrier function. Transfected Caco-2 monolayers with stable expression of Myc-FLAG tagged JAM-A (F11R) or pCMV6 empty vector were incubated in HBBS+ at 37°C for 1-h. FITC-dextran 40-kDa was added to the apical compartments of the transwell plates, followed by treatment of both apical and basal compartments with 0% (HBSS+ alone) or 6% v/v ethanol. TEER was measured immediately and again after 3-h incubation. Expression of the tagged-JAM-A in the differentiated monolayers was confirmed by western blot (A). TEER values (B), and FITC-dextran concentration (C), of basal solutions after 3-h incubation are reported as means ± SEM. Western blot representative of two independent experiments. All other data are representative of multiple individual monolayer inserts from two independent experiments. Data are analyzed by two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.