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Introduction (max. 800 characters): Lung cells are constantly exposed to stress signals, which can contribute to various diseases. The endoplasmic reticulum (ER) plays a crucial role in protein synthesis and cell stress response, when accumulation of misfolded proteins triggers a general stress signaling pathway - unfolded protein stress response (UPR). However, the exact impact of UPR on the growth, differentiation, and regeneration of lung cells is not fully understood. Expandable lung epithelial (ELEP) cells are derived from human embryonic stem cells and provide a unique model for studying lung biology and regeneration. The proposed hypothesis suggests that ER stress induces significant changes in ELEP cells, such as epithelial-mesenchymal transition (EMT), through increased expression of UPR proteins. Materials and methods (max. 1000 characters):* ELEP cells were differentiated from human embryonic stem cell (hESC) lines as described previously. Briefly, hESCs were differentiated into distal foregut endoderm through the activin signaling. Then lung-specific differentiation was then mediated by FGF7, FGF10, BMPs, and WNT signaling pathways. Retinoic acid and dexamethasone were used for the maturation and acquisition of functional characteristics of the ELEP cells. Acquisition of differentiated phenotype of ELEP cells was confirmed by TTF1 expression by flow cytometry and western blotting. Viability was estimated by determination of the conversion rate of the tetrazolium dye (MTT) to the insoluble formazan. mRNA and protein levels were determined by qRT-PCR and western blotting, respectively. Subcellular localization of major regulators was determined by immunofluorescent microscopy. Results and conclusions (max. 700 characters):* ELEP cells showed high sensitivity to accumulation of misfolded proteins in the ER by N-clycosylation inhibition, and high rate of cell death. Induction of ER stress was confirmed by upregulation of major ER chaperon BiP and downstream transcription factor CHOP. In addition, we observed increase of E-cadherin signal in cytoplasm, suggesting accumulation of misfolded protein the ER,while N-cadherin retained the membrane signal, even upon ER stress. Induction of ER stress also induced changes in cell morphology, from the cobblestone, epithelial towards the elongated, mesenchymal phenotype. In summary, we showed that ER stress in ELEP cells induced EMT through the activation of the UPR pathway. Acknowledgements (max. 200 characters):* This research is supported by the Czech Science Foundation, project no. GA23-06675S, and Masaryk University, project no. MUNI/A/1301/2022.
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