Tunicamycin disrupts Caco-2 monolayer integrity through caspase- and RIPK1-independent cell death that is associated with lysosomal membrane permeabilization.

<p><b>(A)</b> Western Blot for active Caspase-3 in differentiated Caco-2 monolayers after apical stimulation of 10 μg/ml TM for indicated time periods with or without TUDCA pre-treatment. <b>(B)</b> Relative level of DNA fragmentation in differentiated Caco-2 monolayers after indicated time periods of apical stimulation with 10 μg/ml TM, Change in TEER of differentiated Caco-2 monolayers after one hour pre-treatment with <b>(C)</b> TUDCA, cell death inhibitor(s) (50 μM zVAD, 30 μM Necrostatin-1), <b>(D)</b> N-acetyl-cysteine (0.1 mM, 10 mM) or their vehicles (PBS, DMSO) and subsequent apical stimulation with 10 μg/ml tunicamycin during 48 hours. <b>(E)</b> Representative microscopic images of Caco-2 monolayers stained with acridine orange after one hour pre-treatment with TUDCA or PBS and subsequent apical stimulation with 10 μg/ml TM or vehicle for 48 hours. The experiment shown is a representative of two independent experiments. <b>(F)</b> Change in TEER and <b>(G)</b> Representative acridine stainings of differentiated Caco-2 monolayers after one hour pre-treatment with TUDCA or PBS together with glucose or sucrose and subsequent apical stimulation with 10 μg/ml tunicamycin or vehicle during 48 hours. The results shown are representative of two independent experiments and represent the means ± standard deviation of triplicate wells.