Functions of acetylated Klf5 in mouse ESC differentiation.

<p>(A) Expression of total, Ac-, and unAc-Klf5 during ESC differentiation. The nonspecific band in the anti-Klf5 blot is marked by an asterisk. V6.5 ESCs were cultured without LIF and feeder cells. Cells were harvested on day 0, 2, 4, and 6, and subjected to Western blot assay. (B) Klf5 knockdown affects the expression of pluripotency genes and differentiation genes in day 4 EBs. Day 4 EBs from shGFP control ESCs and two independent stable Klf5 knockdown ESC clones were harvested for quantitative RT-PCR analysis. Averages and standard deviations from three independent experiments were plotted. (C) Rescue effect of WT, KR, and KQ Klf5 in <i>Klf5</i> knockdown ESCs. <i>shKlf5</i> targeting sequences in WT, KR, and KQ Klf5 were mutated to render them resistant to <i>shKlf5</i> knockdown. Stable ESC lines overexpressing shKlf5-immune WT, KR, and KQ Klf5 were established in the Klf5 knockdown ESCs. Day 4 EBs from these ESCs were harvested for quantitative RT-PCR analysis. Averages and standard deviations from three independent experiments were plotted. P values were calculated by reusable two-factor analysis of variance.