Sustained PGC-1α loss of function triggers mesenchymal transition.

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Sustained PGC-1α loss of function triggers mesenchymal transition. Sustained PGC-1α loss of function triggers mesenchymal transition. (A) Immunodetection of the tight junction protein ZO-1 (red) in shPGC1A and shCtrl ARPE-19 cells matured for up to 21 d. Scale bar is 10 μm. (B) Barrier integrity measured by TER (n = 3 per condition). (C) Immunostaining of the vimentin IFs (red) in shPGC1A and shCtrl ARPE-19 cells matured from 7 up to 21 d. DAPI (blue) was used to stain nuclei. Scale bar is 50 μm. (D) Expression of EMT-related genes ZEB1, ZEB2, VIM, TWIST, and the cell cycle regulator p53 measured by qPCR analysis (n = 3–4). (E–H) Protein levels of the EMT transcriptional factors ZEB1 (E) and TWIST1 (F), the mTOR-dependent phospho-S6 ribosomal protein (Ser235/236) (G) and total AMPKα (H) in shPGC1A and shCtrl cells at 21 d. Immunoblots are representative of n = 3–4 samples per conditions. (I) Quantification of cell number per well expressed as fold change to shCtrl for each time point demonstrating significant increases in cell density in shPGC1A cells (n = 8–15). Error bars are means ± SEM. Data were analyzed by unpaired t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 compared with their respective shCtrl at each time points. Mariana Aparecida Brunini Rosales et al. LSA 2019;2:e201800212 © 2019 Rosales et al.