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. 2020 Dec 15;22(1):e50535. doi: 10.15252/embr.202050535

Figure 6. NUMB alternative splicing impacts NOTCH activity during EPC generation.

Figure 6

  1. The top 10 Reactome terms enriched from differential splicing events regulated by PLB. To define the differential splicing events, genes with FPKM > 1 in at least one differentiation stage were selected. Among them, the differential splicing events were identified by the double cutoffs of delta PSI > 0.2 and FDR < 0.05.
  2. GSEA analysis of NOTCH signaling in isoform level between DMSO control and 2.5 nM of PLB‐treated cells.
  3. The bar graph representing the percentage of CD31+CD34+ cells at day 5 of differentiation after treatment with DMSO and various concentrations of DAPT starting from day 2.5.
  4. The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2‐APLNR+ cells, day 5‐DMSO–treated, and day 5‐PLB‐treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO‐ and PLB‐treated cells. The heatmap was scaled with Z‐Score using the log2(FPKM) expression of indicated genes. n = 2 technical replicates
  5. RT–qPCR assay showing the expression of HES1 mRNA in the GFP+ cells on day 5 of differentiation with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5.
  6. RT–qPCR analysis confirmed the overexpression of HES1 mRNA after DOX induction. The ACTB gene was used as a control. The data were normalized to the mRNA level in empty vector control cells.
  7. Western blotting showing the level of HES1 overexpression after DOX induction. The GAPDH gene was used as a loading control.
  8. The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression.
  9. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).
  10. A hypothetical model. During hematopoietic differentiation from hESCs, a splicing factor switch occurs during the transition from mesoderm APLNR+ cells to EPCs. Concomitant with the switch of components in the splicing machinery, the splicing regulator SRSF2 is downregulated, leading to the enrichment of an EPC‐induced NUMB_S isoform. Alternative splicing of NUMB controls NOTCH activity, probably by suppressing HES1 expression and, ultimately, regulates EPC specification. The blue and red colors represent the two switching clusters within splicing factors during hematopoietic differentiation.

Data information: Results given are mean ± SD. P‐values were determined by an unpaired two‐tailed Student’s t‐test in (C), (E), (F), and (I). **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.

Source data are available online for this figure.