Figure 5.

MiR-29b Acts Downstream of Pou3f1 to Drive Mouse ESCs into the NTE Lineage

(A) Luciferase reporter assay in 3T3 cells transfected control (pcDNA3.1), Sox1, Sox2, Pax6, Zfp521, Pou3f1, and Zic1, respectively, with pGL3-miR-29b promoter.

(B and C) qPCR (B) and western blotting (C) in tet-on shPou3f1 cell line with and without Dox showed that Pou3f1 knockdown reduced the expression of miR-29b and increased the protein level of DNMT3A. The protein abundance of POU3F1 and DNMT3A was quantified with normalization by signals of GAPDH.

(D) Luciferase reporter assay in 3T3 cells transfected control (pcDNA3.1) and Pou3f1, respectively, with pGL3 fragments of miR-29b promoter.

(E) ChIP assay showed enrichment of POU3F1 at the promoter region of miR-29b.

(F and G) Western blotting showed increased level of POU3F1 and decreased level of DNMT3A in tet-on Pou3f1 cell line with and without Dox on D3 of NTE (F) and NCC (G) differentiation. The protein abundance of POU3F1 and DNMT3A was quantified with normalization by signals of GAPDH. Overexpression of Pou3f1 upregulates the expression level of miR-29b on D3 of NTE (F) and NCC (G) differentiation, as verified by qPCR.

(H–J) Inhibiting miR-29b offset the promotion of NTE differentiation by Pou3f1, as shown by qPCR (H), FACS (J), and immunofluorescence (I) as well as the quantification of SOX2-positive cells on D5 of NTE differentiation EBs.

(K and L) Inhibiting miR-29b rescue the defection of NCC differentiation by Pou3f1, as shown by qPCR (K) and immunofluorescence as well as quantification of P75-positive cells (L) on D12 of NCC differentiation. The dashed lines indicate the region of the sphere.

Means ± SEM of n = 3 independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus the control; ^#p < 0.05, ^##p < 0.01 versus the fragment-B group (D) or tet-on Pou3f1 + Dox group (H–L). Scale bars, 100 μm.