Figure 3: G1 arrest facilitates fibroblast-to-iDA conversion.

(a) The percentage of EdU-labelled cells after Dox induction of the indicated reprogramming factors for 0, 24 and 48 h. *P<0.01, unpaired, two tailed Student’s t-tests versus 0 h; n=6 wells from 3 independent experiments for each condition. (b–d) The percentage of EdU⁺ cells in all DAPI⁺ cells (b), the percentage of EdU⁺Tuj1⁺ double-positive cells in all DAPI⁺ cells (c) and the percentage of EdU⁺TH⁺ double-positive cells in all DAPI⁺ cells (d) at day 9 of reprogramming with the indicated factors. *P<0.05, unpaired, two tailed Student’s t-tests versus ANL or ANLm; n=6 wells from 3 independent experiments. Representative images of cells co-stained for Tuj1/EdU/DAPI (e–h) or TH/EdU/DAPI (i–l) at day 9 of reprogramming with the indicated factors. (m) Improved protocol using serum withdrawal for 24 h in DMEM/F12 to arrest cell cycle at G1 before Dox induction of reprogramming factors in neural induction media. Cell cycle analysis for MRC5 cells (n) or MRC5 cells transduced with ANLmp viruses (o) in the presence or absence of fetal bovine serum (FBS). (p) Percentage of EdU-labelled cells after serum withdrawal and treatment without or with Dox for 24 h for the indicated reprogramming factors. ^‡P<0.05, unpaired, two tailed Student’s t-tests versus the preceding bar. *^{, #}P<0.05, unpaired, two tailed Student’s t-tests versus ANL without or with Dox, respectively; n=6 wells from 3 independent experiments. (q) TH⁺ and Tuj1⁺ neurons generated with the protocol in m. Lack of EdU labelling in Tuj1⁺ neurons (r) and TH⁺ neurons (s) derived from the protocol in m. Pulse labelling of EdU (for 2 h) was performed at 24 h in Dox treatment. Staining was done at day 10. Scale bars, 100 μm.