Monitoring autophagic flux using p62/SQSTM1 based luciferase reporters in glioma cells

Zhang, Min; Yao, Ting; Mingtao, Gong; Tang, Xiaofei; Dong, Yan; Zhang, Chenguang; Ding, Wei

doi:10.1016/j.yexcr.2017.12.027

Cited by 24 publications

(20 citation statements)

References 28 publications

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“…The lysates from the two populations (wild type and mutant p62) were compared to monitor the autophagic flux. The performance of this probe was reported to be comparable to GFP-LC3-RFP-LC3ΔG probe described earlier in the review ( Min et al, 2018 ).…”

Section: High Throughput Assays To Monitor Autophagysupporting

confidence: 60%

“…Luciferase based HTS autophagy assay has been reported for mammalian cells as well. In a study by Min et al (2018) , a luciferase variant Luc2p was fused with the wild type p62/SQSTM1 or a deletion version of p62 (p62 lacking the ubiquitin binding domain) and transfected into glioma cells. The lysates from the two populations (wild type and mutant p62) were compared to monitor the autophagic flux.…”

Section: High Throughput Assays To Monitor Autophagymentioning

confidence: 99%

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Chemical Biology Strategies to Study Autophagy

Mishra

Ammanathan

Manjithaya

2018

Front. Cell Dev. Biol.

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Growing amount of evidence in the last two decades highlight that macroautophagy (generally referred to as autophagy) is not only indispensable for survival in yeast but also equally important to maintain cellular quality control in higher eukaryotes as well. Importantly, dysfunctional autophagy has been explicitly shown to be involved in various physiological and pathological conditions such as cell death, cancer, neurodegenerative, and other diseases. Therefore, modulation and regulation of the autophagy pathway has emerged as an alternative strategy for the treatment of various disease conditions in the recent years. Several studies have shown genetic or pharmacological modulation of autophagy to be effective in treating cancer, clearing intracellular aggregates and pathogens. Understanding and controlling the autophagic flux, either through a genetic or pharmacological approach is therefore a highly promising approach and of great scientific interest as spatiotemporal and cell-tissue-organ level autophagy regulation is not clearly understood. Indeed, chemical biology approaches that identify small molecule effectors of autophagy have thus a dual benefit: the modulators act as tools to study and understand the process of autophagy, and may also have therapeutic potential. In this review, we discuss different strategies that have appeared to screen and identify potent small molecule modulators of autophagy.

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Section: High Throughput Assays To Monitor Autophagysupporting

confidence: 60%

Section: High Throughput Assays To Monitor Autophagymentioning

confidence: 99%

Chemical Biology Strategies to Study Autophagy

Mishra

Ammanathan

Manjithaya

2018

Front. Cell Dev. Biol.

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“…In EBSS-treated cells, the number of RFP + GFP À puncta was greater than the number of RFP + GFP + puncta (Figure 2A), suggesting that complete autophagy flux dominates. Torin1, a potent inhibitor of mTORC1/2, is generally applied as an autophagy inducer (Min et al, 2018). Hydroxychloroquine sulfate (CQ), an acidification inhibitor that impedes lysosome-mediated degradation, can accumulate autophagosomes and suppress autophagic flux (Kou et al, 2017).…”

Section: (Legend On Next Page)mentioning

confidence: 99%

The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses

Wang

Liu

et al. 2019

Cell Reports

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“…Additionally, GANT61 decreased the luciferase activity in lysates from T-HESCs transfected with the Luc2p-p62 plasmid, which expresses a recombinant p62 protein fused with a luciferase variant of Luc2p at the N-terminus to distinguish it from endogenous p62 38 , suggesting that GANT61 accelerated Luc2p-p62 degradation (Fig. 2k).…”

Section: Shh Signaling Negatively Regulates Autophagy Initiationmentioning

confidence: 95%

Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells

et al. 2020

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Autophagy can be dynamically induced in response to stresses and is an essential, ubiquitous intracellular recycling system that impacts the fate of damaged resident cells, thereby influencing wound healing. Endometrial fibrosis is a form of abnormal wound healing that causes intrauterine adhesion (IUA) and infertility. We previously demonstrated that overactivated sonic hedgehog (SHH) signaling exacerbated endometrial fibrosis, but the role of autophagy in this process is still unknown. Here, we report that impaired autophagy participates in SHH pathway-induced endometrial fibrosis. Endometrial stroma-myofibroblast transition accompanied by autophagy dysfunction was present in both endometrial biopsies of IUA patients and Amhr2cre/+R26-SmoM2+/− (AM2) transgenic mouse. Mechanistically, SHH pathway negatively regulated autophagy through pAKT-mTORC1 in a human endometrial stromal cell line (T-HESCs). Furthermore, SHH pathway-mediated fibrosis was partly counteracted by autophagy modulation in both T-HESCs and the murine IUA model. Specifically, the impact of SHH pathway inhibition (GANT61) was reversed by the pharmacological autophagy inhibitor chloroquine (CQ) or RNA interference of autophagy-related gene ATG5 or ATG7. Similar results were obtained from the murine IUA model treated with GANT61 and CQ. Moreover, promoting autophagy with rapamycin reduced fibrosis in the AM2 IUA model to baseline levels. In summary, defective autophagy is involved in SHH pathway-driven endometrial fibrosis, suggesting a potential novel molecular target for IUA treatment.

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Monitoring autophagic flux using p62/SQSTM1 based luciferase reporters in glioma cells

Cited by 24 publications

References 28 publications

Chemical Biology Strategies to Study Autophagy

Chemical Biology Strategies to Study Autophagy

The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses

Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells

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