Dysregulated signal transducer and activator of transcription 3 drives intestinal permeability and may contribute to acute-on-chronic liver failure

Figure 4 Expression of relevant tight junction proteins is significantly modified by differential signal transducer and activator of transcription 3 expression in Caco-2 and T84 monolayers. Monolayers consisting of Caco-2 or T84 cells with different levels of signal transducer and activator of transcription 3 (STAT3) expression were generated and epithelial permeability was determined by transepithelial electrical resistance measurements over 28 days. A and B: Whole protein extracts were analyzed via western blot (an exemplary blot is shown on which the protein extracts from three independently performed test series were applied next to each other; C and D: Quantified using Fiji (ImageJ distribution; version 2.14.0/1.54f). Values were normalized to Caco-2 or T84 control cell lines. Bars indicate mean ± SD. Western blots were performed with specific primary antibodies against STAT3, phospho-STAT3, STAT1, phospho-STAT1, Ocln, Cldn1, Cldn2, Cldn3 and zonulin 1, while β-actin served as a housekeeping protein. As shown, reduced (STAT3 -) as well as enhanced STAT3 (STAT3 +) expression had an important impact on the activation of STAT1 as well as the expression of tight junction proteins. P values were calculated using two-way analysis of variance followed by Dunnett’s multiple comparisons test. The data shown are mean ± SD, and the following P values were considered statistically significant (n ≥ 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. STAT3: Signal transducer and activator of transcription 3; pSTAT3: Phospho-signal transducer and activator of transcription 3; STAT1: Signal transducer and activator of transcription 1; pSTAT1: Phospho-signal transducer and activator of transcription 1; ZO-1: Zonula occludens-1.