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. 2016 Jul 14;7(2):207–219. doi: 10.1016/j.stemcr.2016.06.008

Figure 4.

Figure 4

miR-9/9∗ Expression in Proliferating lt-NES Cells upon Modulation of Notch Pathway Activity

(A–C) qRT-PCR analyses of HEY1 mRNA (A), miR-9 (B), and miR-9∗ (C) in I3 lt-NES cells transduced with lentiviral vectors overexpressing in a doxycycline-dependent manner GFP, NICD, or DN-MAML1 after 4 days of doxycycline treatment in the presence or absence of DAPT. Data are normalized to 18S rRNA (A) and miR-16 (B, C) reference levels and presented as average changes + SEM relative to expression in GFP-expressing I3 lt-NES cells (GFP, equal to 1; n ≥ 4; ∗p ≤ 0.05, ∗∗p ≤ 0.01, Student's t test).

(D) qRT-PCR analyses of miR-9, miR-9∗, and miR-125b in I3 lt-NES cells transduced with lentiviral vectors overexpressing in a doxycycline-dependent manner GFP, N2ICD, or DN-MAML1 after 4 days of doxycycline treatment. Data are normalized to RNU5A reference levels and presented as average changes + SEM relative to expression in GFP-expressing lt-NES cells (GFP, equal to 1; n = 3; ∗p ≤ 0.05, ∗∗p ≤ 0.01, Student's t test).

(E) Representative images of PCR analyses for expression of the three pri-miR-9 forms (pri-9_1, pri-9_2, and pri-9_3) in I3 lt-NES cells (NES) and fetal brain RNA (FB). 18S rRNA levels were used as loading control.

(F) qRT-PCR analyses of pri-miR-9_2 transcript levels (pri-9_2) in the samples described above. Data are normalized to 18S rRNA reference levels and are presented as average changes + SEM relative to expression in GFP-expressing I3 lt-NES cells (GFP, equal to 1; n ≥ 4; ∗p ≤ 0.05, ∗∗p ≤ 0.01, Student's t test).

(G) Representative images of PCR analyses of the predicted RBPj binding sites in the genomic regions 10 kb upstream of pre-miR-9_2 and pre-miR-125b_2 from immunoprecipitates with antibodies against FLAG (FLAG-tagged NICD) and RBPj compared with IgG negative control (n = 3).