Figure 3.
Overexpression of miR-342-5p Repressed the Formation of Both Suspended Neurospheres and Adherent Colonies, and Repressed the Proliferation of NS/PCs
(A) Single-cell suspensions were prepared from the GE of E15.5 embryos and cultured on poly-L-lysine (PLL)-coated dishes in DF12 medium supplemented with N2, 20 ng/mL bFGF, and 20 ng/mL EGF. After 12 hr, NS/PCs were transfected with 50 or 100 nM miR-342-5p mimics or control oligonucleotides (Ctrl). Cells were detached 4 hr later and cultured under neurosphere conditions for 7 days. The cultures were photographed and the number of neurospheres was determined under a microscope. The transfections were done five times independently.
(B and C) NS/PCs derived from normal embryos were transfected with 100 nM miR-342-5p mimics or control as in (A) (five independent transfections performed). Cells were then replated at clonal density on PLL-coated dishes for 7 days. The number of colonies (B) and cell number per colony (C) were determined.
(D–I) NS/PCs were transfected with miR-342-5p mimics and the control, and were cultured adherently at clonal density for 7 days (D–G) (five independent transfections performed) or at normal density for 48 hr (H and I) (five independent transfections performed). BrdU was added into the medium 18 hr before the end of the experiment. Cells were fixed and stained with anti-BrdU and anti-NESTIN antibodies by immunofluorescence staining (D) or with anti-BrdU, anti-NESTIN, and anti-Ki67 antibodies (H). Nuclei were counterstained with a Hoechst stain. The percentages of BrdU+ (E), NESTIN+ (F), and NESTIN+ BrdU+ (G) cells within one colony were determined (50 colonies counted in each group). The percentages of NESTIN+ BrdU+ cells and NESTIN+ Ki67+ cells within one field were determined (I) (ten fields counted in each group).
Bars, means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.