Volume 23, Issue 1, Pages 220-226 (January 2016) Transient Activation of Mitoflashes Modulates Nanog at the Early Phase of Somatic Cell Reprogramming  Zhongfu Ying, Keshi Chen, Lingjun Zheng, Yi Wu, Linpeng Li, Rui Wang, Qi Long, Liang Yang, Jingyi Guo, Deyang Yao, Yong Li, Feixiang Bao, Ge Xiang, Jinglei Liu, Qiaoying Huang, Zhiming Wu, Andrew Paul Hutchins, Duanqing Pei, Xingguo Liu  Cell Metabolism  Volume 23, Issue 1, Pages 220-226 (January 2016) DOI: 10.1016/j.cmet.2015.10.002 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Metabolism 2016 23, 220-226DOI: (10.1016/j.cmet.2015.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Transient Activation of Mitoflashes at the Early Phase of Reprogramming (A) A typical mitoflash event shown by mito-cpYFP during reprogramming. Mitochondrion #1 shows a sharp increase and subsequent decrease of mito-cpYFP fluorescence, which indicates a mitoflash event, while mitochondrion #2 shows stable fluorescence during the time course. Scale bar corresponds to 3 μm. The amplitudes (ΔF/F0) of each mitochondrion were measured over time (lower panel). (B) Images of mitoflashes at days 0, 3, 5, and 8 during reprogramming (upper panel). Scale bar corresponds to 10 μm. The ΔF/F0 of each indicated mitochondrion (white arrow) was measured over time (lower panel). (C) The ΔF/F0 of mitoflashes were quantified at days 0, 3, 5, and 8 (data are mean ± SEM; n ≥ 12; ∗∗p < 0.01; ∗∗∗p < 0.001). (D) Frequencies of mitoflashes in SKOM infected cells were quantified at days 0, 3, 5, and 8 (data are mean ± SEM; n ≥ 34; ∗p < 0.05; ∗∗∗p < 0.001). (E) The frequency of mitoflashes at day 3 was much higher than day 0 in SKOM retrovirus-infected cells. Surface plots generated using ZEN 2010 software (Carl Zeiss) show mitoflashes in SKOM D0 (upper) and SKOM D3 (lower) infected cells. See also Figure S1 and Movie S1. Cell Metabolism 2016 23, 220-226DOI: (10.1016/j.cmet.2015.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Mitoflash Activation during the Early Phase Enhances Reprogramming (A) Frequencies of mitoflashes in paraquat and mastoparan treated cells were quantified (data are mean ± SEM; n ≥ 31; ∗∗p < 0.01; ∗∗∗p < 0.001). (B) The GFP+ colonies number was determined with paraquat or mastoparan treatment for 8 days or 13 days (data are mean ± SEM; ∗p < 0.05; ∗∗p < 0.01). (C) Frequencies of mitoflashes in CypD overexpressed cells were quantified (data are mean ± SEM; n ≥ 44; ∗p < 0.05). (D) The GFP+ colonies number was determined when CypD was overexpressed. CypD expression for 4 or 13 days (data are mean ± SEM; ∗p < 0.05; ∗∗p < 0.01). (E) The GFP+ colonies number was determined when MnTMPyP, CypD shRNA or siRNA was treated (data are mean ± SEM; ∗p < 0.05; ∗∗p < 0.01). See also Figure S2. Cell Metabolism 2016 23, 220-226DOI: (10.1016/j.cmet.2015.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Mitoflash Activation Enhances Reprogramming Efficiency through Increasing the Expression of Nanog (A) qPCR analysis of pluripotent genes at day 8 of reprogramming (data are mean ± SEM; ∗p < 0.05). (B and C) qPCR analysis of Nanog after paraquat or mastoparan treatment (B) or CypD overexpression (C) at different days (data are mean ± SEM; ∗p < 0.05). (D) Western blot analysis of Nanog after paraquat or mastoparan treatment or CypD overexpression. (E) The GFP+ colonies number with Nanog overexpression was determined with or without paraquat or mastoparan. The cells were treated with paraquat or mastoparan for 8 days. Flag retrovirus-infected cells served as control (data are mean ± SEM; ∗p < 0.05; ∗∗p < 0.01). (F) The GFP+ colonies number was determined when Nanog was overexpressed with or without induced expression of CypD until day 4. Flag retrovirus-infected cells served as control (data are mean ± SEM; ∗∗∗p < 0.001). See also Figure S3, Table S1, and Table S2. Cell Metabolism 2016 23, 220-226DOI: (10.1016/j.cmet.2015.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Mitoflash Activation Decreases the Methylation Status of the Nanog Promoter through Tet2 Occupancy (A and B) The methylation status of the Nanog promoter was analyzed after paraquat or mastoparan treatment at day 7 (A), or CypD overexpression at day 3 (B). An open circle represents an unmethylated CpG, and a solid circle represents a methylated CpG. Each horizontal line represents an individual allele, which contains six CpG sequences. The complete demethylated Nanog promoter was indicated in red frames. (C) ChIP-qPCR analysis of Tet2 binding to the Nanog promoter during reprogramming (control, mastoparan, and paraquat at day 8; Flag and CypD at day 4) (data are mean ± SEM; ∗p < 0.05). (D) Scheme showing the increased frequency of mitoflashes in the early phase of reprogramming. See also Figure S4. Cell Metabolism 2016 23, 220-226DOI: (10.1016/j.cmet.2015.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions