Figure 4.
Flcn Regulates Subcellular Localization of Tfe3
(A) Control (ctrl.1), Flcn shRNA (Flcn.4), and Tfe3 shRNA (Tfe3.3) cell lines were stained for Tfe3 and DNA in 2i conditions (left panel) and 24 hr after inhibitor withdrawal (right panel). Tfe3 was detected in the nucleus (arrowhead) and cytoplasm (open arrowhead).
(B) Cytoplasmic (C) and nuclear (N) fractions of control and Flcn shRNA cells probed with indicated antibodies (open arrowheads indicate Fnip1 and Flcn bands; both bands recognized by Tfe3 antibodies are specific and likely represent phosphorylation variants; Hong et al., 2010).
(C) Box and whisker plots of nuclear/cytoplasmic Tfe3 ratios in ESCs transfected with indicated siRNAs in 2i conditions and 24 hr after inhibitor withdrawal. Indicated cell numbers (white) from three experiments with two ESC lines were quantified. (∗∗) and (∗) indicate Student’s t test values < 1 × 10−100 and 1 × 10−50, respectively.
(D–F) Tfe3 is localized to the nucleus (arrowhead) at E3.5 (D). At E4.5, Tfe3 is found in the nucleus and cytoplasm of Nanog-positive epiblast cells (arrowhead) but stays nuclear in GATA4-positive (open arrowhead) primitive endodermal cells (E). At E5.5, Tfe3 is enriched in the cytoplasm (arrowhead) of Oct4-positive epiblast cells and remains nuclear (open arrowhead) in extraembryonic endoderm cells (F).
See also Figure S4.