JCS02955 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Transgene expressions using retroviral infection. Neural precursor cells, derived from the cortex, LGE, and midbrain at E14, were infected with retroviral constructs pNurr1-IRES-eGFP (A-D), pMash1 (E,F,I), and pNgn2 (G,H,I) at day 2 of in vitro proliferation. Two days after infection, immunocytochemical (A-H) and semi-quantitative RT-PCR (I) analyses were performed. A-H, representative images for GFP (A,B), Nurr1 (C,D), Mash1 (E,F), and Ngn2 (G,H)-immnostainings obtained from the cortical precursor cells. Bar, 10 μm.

• Supplemental Figure 2 -

  Fig. S2. Effects of proneural bHLHs in Nurr1-induced DA differentiation from cultured LGE and midbrain precursor cells. Neural precursor cells were isolated and cultured from embryonic LGE and midbrain at E13, and were transduced with Nurr1 plus Mash1, Ngn1, Ngn2, NeuroD or LacZ (control) using retroviral infection. TH immunocytochemistry was carried out at day 3 of in vitro differentiation. A-D, Mash1-induced morphologic maturation of Nurr1-TH cells in the cultures from embryonic LGE (A,B) and midbrain (C,D). E-N, Inhibitory activities of the atonal-related bHLH proteins on Nurr1-induced TH+ cell yield. Note that Mash1-induced TH+ cell maturation and inhibitory roles of Ngn1, 2, and NueroD are similarly observed in the LGE and midbrain cultures. Bar, 10μm. Insets, DAPI nuclear staining of the same fields.

• Supplemental Figure 3 -

  Fig. S3. Ngn1-mediated repression of TH expression is not attributable to the reduced cell proliferation. (A) Scheme of the experiment. Cultured cortical precursor cells were incubated with Nurr1-retroviruses for 2 hours and then with LacZ- (for control, PD98059-, and SU5402-treated groups) or Ngn1-viruses (for Ngn1-infected group). After 2 hours of incubation, cell differentiation was induced in N2. PD98059 (25 μM) or SU5402 (20 μM) was added to the specified cultures. Six hours later, media were changed with N2. At this point, BrdU-incorporation assay was carried out. Immunocytochemistry for TH/Nurr1 was performed after 1 day of additional culture in N2. (B) Percentage BrdU-positive cells. BrdU (10 μM) was applied to cultures for 40 minutes and followed by anti-BrdU staining. (C) Percentage TH+ cells out of Nurr1+ cells. * Significantly different from the control (Nurr1 + LacZ) at p<0.001. ^# Significantly different from Ngn1-infected cultures (Nurr1 + Ngn1) at p<0.001.