Fig. 4: Zyxin overexpression supresses pluripotency makers.

a, b mCherry vector control (ctrl) and mCherry-Zyxin (zyxin O/E) were transiently transfected into (a) D3 and (b) E14 cells with an increasing amount of plasmid DNA—0.1, 0.2, 0.5, 1.0, and 2.0 μg. The levels of pluripotency markers (Oct4 and Nanog) were analyzed by western blot. Zyxin overexpression was confirmed by detecting with both zyxin and mCherry antibodies. β-tubulin was used as loading control. c Densitometric analysis of pluripotency markers (Oct4 and Nanog) and zyxin was conducted and plotted for D3 and E14. Values were normalized against β-tubulin and presented as fold change using mCherry-control as the internal reference (N = 3). d Changes at transcript levels were analyzed through Real-Time PCR, using primers specifically targeting Oct4, Nanog and Zyxin. Relative quantification values were normalized against β-tubulin and presented as fold change using mCherry-control as the internal reference (N = 3). All results were from at least three independent experiments. Two-tailed unpaired Student’s t-test was used to test the differences between the control and the highest concentration (2.0 μg) of mCherry-Zyxin (zyxin O/E). Error bars represent standard deviations. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.