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. 2020 Oct 5;21(11):e50283. doi: 10.15252/embr.202050283

Figure 4. Exons 1–3 of NDIME antagonize EZH2‐mediated epigenetic silencing of Mef2c.

Figure 4

  • A
    RNA secondary structure was predicted based on MFE and partition function through RNAfold WebServer (left panel, and the color scale represents positional entropy). The thermodynamic ensemble of RNA structures (right panel).
  • B
    RIP analysis using EZH2 antibody for the co‐transfection of Flag‐EZH2 and NDIME exons 1–3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG.
  • C
    Verification of the efficiency of overexpression of exons 1–3 or exon 4 of NDIME in NDIME‐knockout cells at day 5 of neural differentiation via qRT–PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME −/−, NDIME −/−+Rs26‐Exon123, and NDIME −/−+Rs26‐Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram.
  • D, E
    FACS (D), expression of neural‐lineage genes (E), in Ctrl, NDIME −/−, NDIME −/−+Rs26‐Exon123, NDIME −/−+Rs26‐Exon4 cells at day 5 of neural differentiation.
  • F
    Microscopy of the control, NDIME −/−, NDIME −/−+Rs26‐Exon123, and NDIME −/−+Rs26‐Exon4 cells at day 5 of neural differentiation. Scale bars, 100 μm.
  • G
    NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 μm.
  • H
    Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation.
  • I
    Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.
Data information: Data in (B and C) are represented as mean ± SEM, n = 3. *P < 0.05, ***P < 0.001 (unpaired two‐tailed Student's t‐test). Data in (D, E and H) are represented as mean ± SEM, n = 4. ***P < 0.001 (one‐way ANOVA with Bonferroni post hoc test). NS—non‐significant.