Microscopic image-based covariation network analysis for actin scaffold-modified insulin signaling

Noguchi, Yoshiyuki; Kano, Fumi; Maiya, Nobuhiko; Iwamoto, Chisako; Yamasaki, Shoko; Otsubo, Yasufumi; Nakatsu, Daiki; Kunishige, Rina; Murata, Masayuki

doi:10.1016/j.isci.2021.102724

Cited by 2 publications

(6 citation statements)

References 41 publications

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“…In contrast, microscopic single-cell analysis enables a more sensitive and precise analysis by examining only the efficiently resealed cells or the correlation between the amount of antibody introduced and the degradation efficiency. We have employed single-cell analysis with immunofluorescence and confocal microscopy in our previous studies, and this technique enabled the successful analysis and phenotyping of various cells, including resealed cells ( Kano et al, 2017 ; Kunishige et al, 2020 ; Noguchi et al, 2021 ). Therefore, we reasoned that single-cell analysis would allow us to detect the effects of Trim-Away more clearly.…”

Section: Resultsmentioning

confidence: 99%

“…Such variations can lower the signal-to-noise ratio of the analysis because the combined results reflect both efficiently and inefficiently resealed cells. In these cases, immunofluorescence and single-cell analysis can be effectively applied to handle the cell-to-cell variation ( Kano et al, 2017 ; Kunishige et al, 2020 ; Noguchi et al, 2021 ). For example, fluorescently labeled dextran has been used as a marker for permeabilization efficiency; this helps eliminate data on non-permeabilized cells and thereby improves the signal-to-noise ratio ( Kano et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis

Kunishige

Murata

Kano

2022

Front. Cell Dev. Biol.

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“Trim-Away” technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis

Kunishige

Murata

Kano

2022

Front. Cell Dev. Biol.

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“…Covariation network analysis was performed as described in a previous paper [ 15 ]. Briefly, the median of each feature value was first calculated for each time point.…”

Section: Methodsmentioning

confidence: 99%

“…Graph clustering was performed as described in a previous paper [ 15 ]. Briefly, the Overlapping Cluster Generator algorithm [ 17 ] was adapted to the estimated covariation network and graph clustering was performed.…”

Section: Methodsmentioning

confidence: 99%

“…In the present study, we used a novel covariation network known as the Protein Localization and Modification‐based Covariation Network (PLOM‐CON) [ 15 ] combined with a transcriptome analysis to identify the mechanism of action of NC114 and its target molecules. We observed that NC114 treatment caused growth arrest in SW480 colorectal cancer cells by downregulating cell cycle‐related gene expression.…”

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confidence: 99%

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Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells

Taguchi,

Nakaya,

Aizawa

et al. 2024

FEBS Open Bio

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The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/β‐catenin pathway, with significant downregulation of both Ser 675‐phosphorylated β‐catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle‐related gene expression, and also induced growth arrest in SW480 and HCT‐116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C‐δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114‐induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and β‐catenin. Consequently, formation of a T‐cell factor‐4 (TCF4)/β‐catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle‐related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

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Microscopic image-based covariation network analysis for actin scaffold-modified insulin signaling

Cited by 2 publications

References 41 publications

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells

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