Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay

Chen, Liang; Leng, Wei; Li, De Zhi; Xia, Hong; Ren, Min; Tang, Qiong; Gong, Qiyong; Gao, Fa Bao; Bi, Feng

doi:10.1038/s41598-017-08358-3

Cited by 5 publications

(3 citation statements)

References 26 publications

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“…Here, we measured the innate behavior of cells under intrinsic pathway of apoptosis detected by luciferase complementary assay. Various biosensors have been established based on the split luciferase strategy due to the production of detectable signal and high quantum yield (Azad et al, 2014;Chen et al, 2017;Leng et al, 2013;Li et al, 2017a;Mohammadi et al, 2017). Specificity and accuracy of measuring is crucial to study a biological process.…”

Section: Discussionmentioning

confidence: 99%

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway

Hosseini

Nikkhah

Hamidieh

et al. 2020

Journal of Cell Science

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Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c. Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro-and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

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Section: Discussionmentioning

confidence: 99%

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway

Hosseini

Nikkhah

Hamidieh

et al. 2020

Journal of Cell Science

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“…The cells were divided into the following groups: (1) pRL‐TK and pGL3‐basic; (2) pRL‐TK, pGL3‐LC3 and shRNA‐NC; (3) pRL‐TK, pGL3‐LC3 and shRNA‐ZFP27; (4) pRL‐TK, pGL3‐LC3 and shRNA‐NC with 100 μg/mL FAC for 12 h; (5) pRL‐TK, pGL3‐LC3 and shRNA‐NC with 100 μM DFO for 12 h. After transfection for 48 h, the cells were plated in 96‐well plates at no less than 2 × 10 4 cells per 75 μL for Dual‐luciferase reporter gene assay. The luciferase activities were measured via Dual‐luciferase assay system (Promega, E2920) according to the manufacturer's instructions using an in vivo bioluminescence imaging system 38 . All assays were replicated for at least three times.…”

Section: Methodsmentioning

confidence: 99%

“…The HEK293T cells were seeded on a 6-well culture plate and transfected with a normalized vector (pRL-TK), a reporter vector tem (Promega, E2920) according to the manufacturer's instructions using an in vivo bioluminescence imaging system. 38 All assays were replicated for at least three times.…”

Section: Luciferase Assaymentioning

confidence: 99%

Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

Wang,

Wen,

Chen

et al. 2023

J Cellular Molecular Medi

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Parkinson's disease (PD) is a challenge because of the ageing of the population and the disease's complicated pathogenesis. Accumulating evidence showed that iron and autophagy were involved in PD. Nevertheless, the molecular mechanism and role of iron and autophagy in PD are not yet elucidated. In the present study, it was shown that PD mice had significant motor dysfunction, increased iron content, less dopamine neurons and more α‐synuclein accumulation in the substantia nigra. Meanwhile, PD mice treated with deferoxamine exhibited less iron content, relieved the dyskinesia and had a significant increase in dopamine neurons and a significant decrease in α‐synuclein. Autophagy induced by LC3 was inhibited in PD models with iron treatment. Following verification showed that iron aggregation restrained insulin‐like growth factor 2 (IGF2) and transcription factor zinc finger protein 27 (ZFP27) in PD models. In addition, LC3‐induced autophagy flux was reduced with ZFP27 knockdown. Furthermore, ZFP27 affected autophagy by regulating LC3 promoter activity. These data suggest that iron deposition inhibits IGF2 and ZFP27 to reduce LC3‐induced autophagy, and ultimately decrease dopamine neurons, accelerating PD progression. Our findings provide a novel insight that ZFP27‐mediated iron‐related autophagy and IGF2 may activate the downstream kinase gene to trigger autophagy in the PD model.

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The F-box protein RhSAF destabilizes the gibberellic acid receptor RhGID1 to mediate ethylene-induced petal senescence in rose

Lu,

Zhang,

et al. 2024

The Plant Cell

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Roses are among the most popular ornamental plants cultivated worldwide for their great economic, symbolic, and cultural importance. Nevertheless, rapid petal senescence markedly reduces rose (Rosa hybrida) flower quality and value. Petal senescence is a developmental process tightly regulated by various phytohormones. Ethylene accelerates petal senescence, while gibberellic acid (GA) delays this process. However, the molecular mechanisms underlying the crosstalk between these phytohormones in the regulation of petal senescence remain largely unclear. Here, we identified SENESCENCE-ASSOCIATED F-BOX (RhSAF), an ethylene-induced F-box protein gene encoding a recognition subunit of the SCF-type E3 ligase. We demonstrated that RhSAF promotes degradation of the GA receptor GIBBERELLIN INSENSITIVE DWARF1 (RhGID1) to accelerate petal senescence. Silencing RhSAF expression delays petal senescence, while suppressing RhGID1 expression accelerates petal senescence. RhSAF physically interacts with RhGID1 s and targets them for ubiquitin/26S proteasome-mediated degradation. Accordingly, ethylene-induced RhGID1C degradation and RhDELLA3 accumulation are compromised in RhSAF-RNAi lines. Our results demonstrate that ethylene antagonizes GA activity through RhGID1 degradation mediated by the E3 ligase RhSAF. These findings enhance our understanding of the phytohormone crosstalk regulating petal senescence and provide insights for improving flower longevity.

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Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay

Cited by 5 publications

References 26 publications

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway

Iron‐inhibited autophagy via transcription factor ZFP27 in Parkinson's disease

The F-box protein RhSAF destabilizes the gibberellic acid receptor RhGID1 to mediate ethylene-induced petal senescence in rose

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