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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front Biol    2010, Vol. 5 Issue (5) : 424-430    https://doi.org/10.1007/s11515-010-0800-4
RESEARCH ARTICLE
Differential effects of recombinant fusion proteins TAT-OCT4 and TAT-NANOG on adult human fibroblasts
Jiani CAO1,2, Zhifeng XIAO1, Bing CHEN1, Yuan GAO1, Chunying SHI1, Jinhuan WANG3, Jianwu DAI1()
1. Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China; 2. Graduate School, Chinese Academy of Sciences, Beijing 100080, China; 3. Institute of Neurosurgery, Tianjin Huanhu Hospital, Tianjin 300060, China
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Abstract

OCT4 and NANOG are two important transcription factors for maintaining the pluripotency and self-renewal abilities of embryonic stem (ES) cells. Meanwhile they play key roles in the induced pluripotent stem (iPS) cells. In this study, recombinant transcript factors TAT-NANOG and TAT-OCT4, which contained a fused powerful protein transduction domain (PTD) TAT from human immunodeficiency virus (HIV), were produced. Each fusion protein could be transported into human adult fibroblasts (HAF) successfully and activated the endogenous transcription of both nanog and oct4. Our study revealed the inter-regulation and autoregulation abilities of solo oct4 or nanog in the process of iPS cell reprogramming. Meanwhile the transduction of TAT-NANOG could accelerate the growth rate of HAF cells, and the key cell cycle regulator cdc25a was up-regulated. Thus cdc25a may be involved in the regulation of cell growth by NANOG. In addition, the TAT fusion protein technology provided a novel way to improve cell growth that is more controllable and safer.
Keywords induced pluripotent stem cells (iPS cells)      OCT4      NANOG      protein transduction domain      TAT      cell proliferation     
Corresponding Author(s): DAI Jianwu,Email:jwdai@genetics.ac.cn   
Issue Date: 01 October 2010
 Cite this article:   
Jiani CAO,Zhifeng XIAO,Bing CHEN, et al. Differential effects of recombinant fusion proteins TAT-OCT4 and TAT-NANOG on adult human fibroblasts[J]. Front Biol, 2010, 5(5): 424-430.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0800-4
https://academic.hep.com.cn/fib/EN/Y2010/V5/I5/424
Fig.1  A: Schematic diagram of the TAT fusion protein expression vector. The His is an affinity tag used to purify the engineered proteins, the respective cDNA of and is connected with TAT and the hemaglutinin (HA) tag at the N-terminus. B: Protein profiles of negative control (Lanes 2 and 5); TAT-OCT4 (Lanes 3 and 6); TAT-NAONG (Lanes 3 and 7); Lane 1: molecular weight markers (kDa); lanes 2–4: total soluble proteins; lanes 5–7: inclusion bodies; C: SDS-PAGE of purified TAT-NANOG and TAT-OCT4; D: Identification of TAT-NANOG and TAT-OCT4 by anti-NANOG and anti-OCT4 antibodies respectively.
Fig.1  A: Schematic diagram of the TAT fusion protein expression vector. The His is an affinity tag used to purify the engineered proteins, the respective cDNA of and is connected with TAT and the hemaglutinin (HA) tag at the N-terminus. B: Protein profiles of negative control (Lanes 2 and 5); TAT-OCT4 (Lanes 3 and 6); TAT-NAONG (Lanes 3 and 7); Lane 1: molecular weight markers (kDa); lanes 2–4: total soluble proteins; lanes 5–7: inclusion bodies; C: SDS-PAGE of purified TAT-NANOG and TAT-OCT4; D: Identification of TAT-NANOG and TAT-OCT4 by anti-NANOG and anti-OCT4 antibodies respectively.
Fig.2  Cells were also stained with PI to visualize the nuclei (Red) and the images were merged. Bars= 50 μm. B: The transcript activity of TAT fusion protein detected by the reporter assay. The average of the three independent experiments is presented, and the error bars are shown.
Fig.2  Cells were also stained with PI to visualize the nuclei (Red) and the images were merged. Bars= 50 μm. B: The transcript activity of TAT fusion protein detected by the reporter assay. The average of the three independent experiments is presented, and the error bars are shown.
Fig.3  Quantitative RT-PCR for the expression of (A) and (B) in TAT-OCT4, TAT-NANOG or PBS (control) treated HAF cells respectively. The data represent the mean±SEM (error bars) from three independent experiments. Both TAT-OCT4 and TAT-NANOG are insufficient to activate the expression of , and . RT-PCR analysis of the pluripotent gene expression in TAT fusion protein treated and control HAF cells, with the human ES cells as positive control (C). The value for the control is set to 1, and all other values are calculated with respect to this. *: <0.05, **: <0.01, ***: <0.001.
Fig.3  Quantitative RT-PCR for the expression of (A) and (B) in TAT-OCT4, TAT-NANOG or PBS (control) treated HAF cells respectively. The data represent the mean±SEM (error bars) from three independent experiments. Both TAT-OCT4 and TAT-NANOG are insufficient to activate the expression of , and . RT-PCR analysis of the pluripotent gene expression in TAT fusion protein treated and control HAF cells, with the human ES cells as positive control (C). The value for the control is set to 1, and all other values are calculated with respect to this. *: <0.05, **: <0.01, ***: <0.001.
Fig.4  Representative morphologies of human adult fibroblasts (HAF) treated with PBS (control), TAT-OCT4 or TAT-NANOG are shown as indicated. Bars= 200 μm. A: Data are presented as means±SD. Results are obtained from three independent experiments (B).
Fig.4  Representative morphologies of human adult fibroblasts (HAF) treated with PBS (control), TAT-OCT4 or TAT-NANOG are shown as indicated. Bars= 200 μm. A: Data are presented as means±SD. Results are obtained from three independent experiments (B).
Fig.5  was up-regulated in TAT-NANOG treated HAF cells. RT-PCR for the expression of (A) and (B) in PBS (control), TAT-NANOG or TAT-OCT4 treated human adult fibroblasts (HAF) respectively. The data represent the mean±SEM (error bars) from three independent experiments. The value for the control is set to 1, and all other values are calculated with respect to this. *: <0.05, **: <0.01, ***: <0.001.
Fig.5  was up-regulated in TAT-NANOG treated HAF cells. RT-PCR for the expression of (A) and (B) in PBS (control), TAT-NANOG or TAT-OCT4 treated human adult fibroblasts (HAF) respectively. The data represent the mean±SEM (error bars) from three independent experiments. The value for the control is set to 1, and all other values are calculated with respect to this. *: <0.05, **: <0.01, ***: <0.001.
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