Functions of Ac-Klf5 in self-renewal and pluripotency regulation.

<p>(A) The effect of WT, KR, and KQ Klf5 overexpression on the expression of pluripotency genes in mouse ESCs. Empty pCAGIPuro, Flag-tagged WT, KR and KQ Klf5 overexpression plasmids were transfected into V6.5 ESCs with Lipofectamine 3000. Two days after transfection, cells were harvested and subjected to RNA purification or Western blot. The top panel shows RNA quantification results (averages and standard deviations from three independent experiments were plotted), and the bottom panel shows Western blots to demonstrate the similar expression levels of Flag-tagged WT, KR and KQ Klf5. (B) The effect of WT, KR, and KQ Klf5 overexpression on the expression of pluripotency genes during mouse ESC differentiation. Experiments were carried out as described in (A), except for that LIF was withdrawn from the medium upon transfection. Averages and standard deviations from three independent experiments were plotted. (C) Relative expression level of total Klf5 mRNA in control, WT, KR, and KQ Klf5 overexpression ESC lines. Averages and standard deviations from three independent experiments were plotted. (D) Proliferation rates of control, WT, KQ, and KR Klf5 overexpression ESCs. 1.5×10⁵ Cells were seeded in one well of a 6-well plate, and 48 hours later, total cell number was counted to measure the proliferation rate. Averages and standard deviations from three independent experiments were plotted. P values were calculated by reusable two-factor analysis of variance. (E) Colony forming assays for control, WT, KR, and KQ Klf5 overexpression ESCs growing without LIF and feeder cells. The left panel shows the image of AP stained colonies of each ESC line, and the quantification result of colony number is plotted in the right panel. Averages and standard deviations from three independent experiments were plotted. P values were calculated by reusable two-factor analysis of variance. (F) Reprogramming ability of WT, KR, and KQ Klf5 in combination with Oct4, Sox2 and c-Myc. MEFs were reprogrammed by WT, KQ, or KR Klf5, together with Oct4, Sox2, and c-Myc. Accumulated iPS colony numbers of three independent experiments (labeled with three different colors) are shown in the plot.