Fig. 5. ZEB1 positively regulates the mitochondrial respiration and aerobic glycolysis mediated EMT of OSCC.

A Wound healing assays were used to evaluate the expression of ZEB1 in regulating the migratory ability of HN6 and Cal27 cell lines. B, C Transwell invasion assays in ZEB1-knockdown cells and ZEB1-overexpressing cells. Median values are plotted in the column with data generated from at least three technical replicates, statistical analysis was performed by unpaired t-tests. D, E Zebrafish xenografts revealed that the knockdown of ZEB1 inhibited the cells invasion ability, whereas the overexpression of ZEB1 promoted the invasion ability of oral cancer cell lines. Median values are plotted in the column, statistical analysis was performed by unpaired t-tests. F WB analysis showing the mitochondrial-related proteins (SIRT3, CTP, SOD1, Citrate Synthase) expression in ZEB1-overexpression cells. G, H Flow cytometry analysis of ROS level (MitoSOX) in HN6 and Cal27 cells. Median values are plotted in each dots plot, the data were generated from four technical replicates, and the statistical analysis was performed by unpaired t-tests I, J ZEB1-knockdown and ZEB1-overexpression HN6 and Cal27 cells were detected for ECAR to indicate the glycolysis stress. K, L ZEB1-knockdown and ZEB1-overexpression HN6 and Cal27 cells were detected for OCR to indicate the mitochondrial respiration. M–O Transmission electron microscope exposing the ultrastructure of mitochondrial in HN6 and Cal27 cells. Median values are plotted in the column with data generated from four technical replicates, statistical analysis was performed by unpaired t-tests (KD knockdown, NC negative control, OE overexpression, CS Citrate Synthase. The data was presented as mean ± SD, *P < 0.05, **P < 0.01. Each dot showed in the dot plots represents the average of at least three independent biological replicates).