Fig. 1: LIN28A expression profile and localization in undifferentiated and differentiated ESCs.

Expression profile of LIN28A in mouse ESCs induced to differentiate into EpiLCs (2 days of differentiation), neural precursors (SFEBs, 4 days of differentiation), neurons (neur. diff., 12 days of differentiation) (A), and mesodermal (4 and 12 days of differentiation) (B) and endodermal (4 and 8 days of differentiation) (C) lineages. The quantification of three independent differentiation experiments is reported in Supplementary Fig. 1D, G. The graphs under the blots represent the relative mRNA expression (using real-time RT-PCR) of the following specific markers: Oct4 for ESCs (also used as a marker for EpilCs), Sox1 for neural precursors, Map2 for neurons, Mlc2v and Foxa2 for mesodermal and endodermal derivatives, respectively. Data are expressed as the mean ± SD of three biological replicates (n = 3), relative to the expression levels in ESCs (Oct4), SFEBs (Sox1), 12 days of neural differentiation (Map2), 12 days of mesodermal differentiation (Mlc2v), or 8 days of endodermal differentiation (Foxa2). Asterisks represent statistical significance based on the Student’s t test (*p ≤ 0.05, **p ≤ 0.005). D Localization of endogenous LIN28A by immunofluorescence in undifferentiated ESCs and sectioned SFEBs. Oct4 and Sox1 were used as markers of undifferentiated ESCs and neural precursors, respectively. Scale bars: 50 μm. DAPI was used to counterstain the nuclei. Magnified images of cropped areas of LIN28A staining are shown. E Cell fractionation followed by western blot was performed to evaluate the distribution of LIN28A in cell compartments of undifferentiated ESCs and SFEBs. The graph shows the mean of LIN28A levels ± SD relative to the quantity measured in the total lysate and normalized against GAPDH for the cytoplasmic fraction (CF) and against histone H3 for the total nuclear fraction (NF). n = 5 biological replicates, *p ≤ 0.05 (Student’s t test).