Fig. 4: LIN28A binds to the PRC2 complex and influences its epigenetic activity.

A Western blot analysis and relative quantification of the H3K27me3 histone mark in WT and Lin28a KO cells in both the undifferentiated and differentiated states. Histone H3 and GAPDH were used as loading control. The use of an antibody against LIN28A confirmed the absence of protein in Lin28a KO cells. The graph shows the mean of the relative quantification ± SD of from six independent biological replicates (n = 6). **p < 0.005 (Student’s t test). B Western blot analysis and relative quantification of different histone marks in WT and Lin28a KO cells. Histone H3 and GAPDH were used as loading controls. The graph shows the mean of the relative quantification ± SD for six biological replicates of H3K4me3 and three biological replicates of H2AK119ub, H3K9me3 and H3K27ac. Student’s t test: ns (not significant). C Western blot analysis and relative quantification of H3K27me3 levels in WT and KO ESCs and SFEBs upon ectopic expression of LIN28A. WT and Lin28a KO cells were transfected with the vector encoding a FLAG-tagged form of LIN28A or the empty vector (Mock) and then differentiated into SFEBs. The LIN28A antibody detects both the endogenous protein (white arrow) and the FLAG-tagged form (black arrow). GAPDH and histone H3 were used as loading controls. The graphs report the mean of the relative quantitation ± SD of four independent (n = 4) biological replicates. *p ≤ 0.05; **p ≤ 0.005 (Student’s t test). D Western blot analysis and relative quantification of pluripotency (OCT4) and differentiation (SOX1) markers in WT and Lin28a KO SFEBs treated with the PRC2 inhibitor, GSK126 during ESCs differentiation toward the neural lineage through SFEB formation (4 days of differentiation). GAPDH and histone H3 were used as loading controls. n = 3 biological replicates. Mean ± SD is reported in the graph. *p < 0.05 (Student’s t test). E Immunofluorescence analysis of whole WT and Lin28a KO SFEBs treated or untreated (NT) with the PRC2 inhibitor GSK126. WT and Lin28a KO ESCs were induced to differentiate into SFEBs for 4 days in the presence or absence of GSK126. Staining for the pluripotency marker OCT4 and the neural differentiation marker SOX1 is shown. Images were generated using the Maximum Projection function (see “Methods”). The graph shows the number of positive cells (mean ± SD) quantified from 50 SFEBs across three independent biological replicates. Statistical significance was assessed using a two-tailed, unpaired Student’s t-test: *p ≤ 0.05. ***p ≤ 0.0001.