Figure 4
Oct4 /Nanog regulates EMT by activating Stat3 pathway. (A) Stat3, p-Stat3 (Y705), and p-Stat3 (S727) protein expression in 97 L-Ctrol cells and 97 L-ON cells. 97 L-ON cells expressed significantly higher amounts of stat3 and p-Stat3 (Y705) than 97 L-Ctrl cell lines. (B) Stat3, p-Stat3Y-705 (total protein and nucleus protein) expression in 97 L-ON-Scramble, 97 L-ON-shOct4, 97 L-ON-shNanog, 97 L-ON-double knockdown cells. (C) Expression of Stat3, p-Stat3 (Y705) proteins, Jak1, p-Jak1, Jak2, p-Jak2 in 97 L-Ctrol, 97 L-shOct4, and 97 L-Oct4 cells. (D) Effect of Nanog expression on stat3 expression, activation, and translocation. Western blot analysis revealed that Nanog had no effect on Stat3 protein, total p-Stat3 (Y705), and p-Jak1, p-Jak2, p-Scr protein expression but increased p-Stat3 (Y705) amounts in nucleus, which indicated nuclear translocation of phospho-Stat3 induced by Nanog. (E) Immunostaining of Nanog (Alexa Fluor®488-labeled anti-Nanog, green color) and p-Stat3 (Alexa Fluor®568-labeled anti-Nanog, red color). Colocalization of Nanog and p-Stat3 (Y-705) is shown as yellow in the merged image. (F) Overexpression of Nanog promoted p-Stat3 nuclear translocation in immunofluorescence analysis. p-Stat3 (Y-705) was localized in cytoplasm and nucleus in 97 L-Ctral cells. After overexpression of Nanog, staining of p-Stat3 protein (green) was significantly increased in the nucleus of 97 L-Nanog cells.