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. Author manuscript; available in PMC: 2014 Jan 19.
Published in final edited form as: Nat Commun. 2013;4:2183. doi: 10.1038/ncomms3183

Figure 4. Neuronal conversion from human fibroblasts does not require long-term expression of NGN2.

Figure 4

(a) qRT-PCR analysis of retrovirus-expressed NGN2 (vNGN2) and the induction of endogenous NGN2 (endoNGN2) at the indicated time points after retroviral transduction of human fetal fibroblasts (means ± s.e.m., n=3 at each time point). Samples from normal adult human brain (Brn) and spinal cord (SC) were used as controls (n=1). (b) Experimental design using doxycycline (Dox) inducible expression system. Cells (5,000/well in 48-well plate) were cotransduced with lentivirus constitutively expressing rtTA3G and Dox-inducible lentivirus(iLV) expressing NGN2-IRES-GFP under the TRE3G promoter. One day post transduction, Dox (0.1 μg/ml) was applied for the indicated periods of time and then withdrawn. The transduction efficiency was about 8% indicated by initial GFP expression. Tuj1-expression was analyzed 35 days later. (c) qRT-PCR analysis of exogenous NGN2 expression, which returns to basal level 6 days after Dox withdrawal (n=1). (d) NGN2 expression is no longer required after neuronal conversion at 21 dpi, although it increases total number of converted neurons at earlier time points. Tuj1+ cells were quantified from a whole well from triplicate samples (means ± s.e.m., n=3). *p=0.04, **p=0.003, and ***p=0.0003 by Student’s t-test.