Figure 1.

KLF4 Nuclear Export Occurs as ESCs Exit Naive Pluripotency

(A) Immunofluorescence of ESCs cultured with LIF/2i and 2, 6, 12, and 24 hr after LIF/2i removal display KLF4 accumulation in the cytoplasm starting at 6 hr (arrows). Merged images display NANOG or KLF4 in green, RNAPII-S5P in red, and DAPI DNA stain in blue. Scale bars, 10 μm.

(B) Box-and-whisker plots display intensities per nucleus of NANOG (left) and KLF4 (right). Green line indicates the average intensity at each time point and black line indicates the median intensity. Boxes indicate interquartile range of intensity values and whiskers indicate the 10^th and 90^th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicate samples and ≥100 nuclei were quantified for each. Statistical differences are indicated by ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(C) Immunoblot of nuclear and cytoplasmic fractions from ESCs cultured with LIF/2i (0) and 2, 6, 12, and 24 hr after LIF/2i removal. Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF1) reveal purity of the cytoplasmic and nuclear fractions, respectively. KLF4 levels are increased in the cytoplasmic fraction at 6 hr and correspondingly decreased in the nuclear fraction. At 24 hr KLF4 was mainly located in the nuclear fraction.

(D) Quantification of relative intensity levels of KLF4 in immunoblots from three biological replicates. Statistical differences compared with the 0-hr values are indicated by ^∗∗p < 0.01 and ^∗∗∗p < 0.001. Error bars represent standard deviation.

See also Figure S1.