High-Throughput Quantitative Detection of Basal Autophagy and Autophagic Flux Using Image Cytometry

SenthilKumar, Gopika; Skiba, Justin H.; Kimple, Randall J.

doi:10.2144/btn-2019-0044

Cited by 13 publications

(12 citation statements)

References 13 publications

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“…Discovering small molecules that enhance lysosomal functionality can be an effective strategy for targeting metabolic disorders such as obesity and diabetes, as they act as activators of autophagic-turnover 10 , 26 . AO staining is a well-known assay to examine the function and integrity of lysosome, which is also used to evaluate the status of autophagic flux 27 , 28 Correlation of the readout with AO fluorescence was validated via measuring the intensity of cells that are stained with different concentration of the staining (Supplementary Fig. 1a ), and via checking that a positive control indatraline, which enhances lysosomal acidity 29 , increased AO intensity 1.2-fold, and a negative control bafilomycin A1, which inhibits acidic lysosome by perturbing proton channel 24 , decreased AO intensity 0.7-fold (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

et al. 2021

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Disorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

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Section: Resultsmentioning

confidence: 99%

Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

et al. 2021

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“…There are several ways to study autophagy in cells. Among these, acridine orange (AO) staining is one of the acceptable tools to detect autophagic cells through the imaging of acidic compartments ( Thome et al, 2016 ; SenthilKumar et al, 2019 ). Therefore, we detected autophagy in the U87-MG after treatment with the C-10 by AO staining.…”

Section: Methodsmentioning

confidence: 99%

Potential of Novel Methyl Jasmonate Analogs as Anticancer Agents to Metabolically Target HK-2 Activity in Glioblastoma Cells

et al. 2022

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Change in the energy metabolism of cancer cells, which display significant differences compared to normal cells, is a rising phenomenon in developing new therapeutic approaches against cancers. One of the metabolic enzymes, hexokinase-II (HK-II) is involved in glycolysis, and inhibiting the HK-II activity may be a potential metabolic target for cancer therapy as most of the drugs in clinical use act on DNA damage. Methyl jasmonate (MJ) is one of the compounds blocking HK-II activity in cancer cells. In a previous study, we showed that the novel MJ analogs inhibit HK-II activity through VDAC detachment from the mitochondria. In this study, to evaluate the potential of targeting HK-2 activity, through patient cohort analysis, we first determined HK-2 expression levels and prognostic significance in highly lethal glioblastoma (GBM) brain tumor. We then examined the in vitro therapeutic effects of the novel analogs in the GBM cells. Here, we report that, among all, compound-10 (C-10) showed significant in vitro therapeutic efficacy as compared to MJ which is in use for preclinical and clinical studies. Afterward, we analyzed cell death triggered by C-10 in two different GBM cell lines. We found that C-10 treatment increased the apoptotic/necrotic cells and autophagy in GBM cells. The newly developed analog, C-10, was found to be lethal against GBM by the activation of cell death authorities, mostly in a necrotic and autophagic fashion at the early stages of the treatment. Considering that possibly decreased intracellular ATP levels by C-10 mediated inhibition of HK-2 activity and disabled VDAC interaction, a more detailed analysis of HK-2 inhibition–mediated cell death can provide a deep understanding of the mechanism of action on the oncosis/necroptosis axis. These findings provide an option to design clinically relevant and effective novel HK-II inhibitors and suggest novel MJ analogs to further study them as potential anticancer agents against GBM.

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“…Cells were assayed for autophagy using 5 mM Acridine Orange (Sigma) for 30 min after which excess was removed by thorough washing with 1X PBS. This fluorophore appears green when diffuse but is shifted to the red end of the spectrum when accumulated in acidic vesicles 92 . As such, excitation/emission wavelengths of 500/526 nm were used to measure intensity of diffuse acridine orange (non-specific) and 460/650 nm to assess autophagic staining.…”

Section: Autophagymentioning

confidence: 99%

Spatially Resolved Phosphoproteomics Reveals Fibroblast Growth Factor Receptor Recycling-driven Regulation of Autophagy and Survival

Watson

Ferguson

Brady

et al. 2021

Preprint

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SummaryReceptor Tyrosine Kinase (RTK) endocytosis-dependent signalling drives cell proliferation and motility during development and adult homeostasis, but is dysregulated in diseases, including cancer. The recruitment of RTK signalling partners during endocytosis, specifically during recycling to the plasma membrane, is still unknown. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b) recycling, we revealed FGFR signalling partners proximal to recycling endosomes (REs) by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphopeptide enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncovered that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the REs, leading to autophagy suppression and cell survival. This adds to the growing importance of RTK recycling in orchestrating cell fate and suggests a therapeutically targetable vulnerability in ligand-responsive cancer cells. Integrating SRP with other systems biology approaches provides a powerful tool to spatially resolve celllar signalling.

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High-Throughput Quantitative Detection of Basal Autophagy and Autophagic Flux Using Image Cytometry

Cited by 13 publications

References 13 publications

Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

Potential of Novel Methyl Jasmonate Analogs as Anticancer Agents to Metabolically Target HK-2 Activity in Glioblastoma Cells

Spatially Resolved Phosphoproteomics Reveals Fibroblast Growth Factor Receptor Recycling-driven Regulation of Autophagy and Survival

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