The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells

Chen, Simin; Lossio, Claudia Figueiredo; Verbeke, Isabel; Verduijn, Joost; Parakhonskiy, Bogdan; Meeren, Louis Van der; Chen, Pengyu; Zaeytijd, Jeroen De; Skirtach, André G.; Damme, Els J. M. Van

doi:10.1016/j.fct.2021.112590

Cited by 5 publications

(10 citation statements)

References 60 publications

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“…Small fragments of multiples of 180-200 bp in size were observed ( Figure 5C ), which is characteristic of internucleosomal cleavage of nuclear DNA into nucleosomal fragments resulting from the activation of an endonuclease(s) ( Oberhammer et al., 1993 ). Conversely, a previous study in animal cells showed that the application of 4 μM OsRIP1 to HeLa cells did not induce DNA fragmentation ( Chen et al., 2021 ). Furthermore, OsRIP1 preferably accumulated at the plasma membrane of HeLa cells, whereas OsRIP1 was internalized into intracellular vacuoles of plant cells ( Figure 2 ).…”

Section: Discussionmentioning

confidence: 82%

“…OsRIP1 labelling with fluorescein isothiocyanate (FITC) (Sigma-Aldrich) was carried out as described previously ( Chen et al., 2021 ). After incubation with OsRIP1-FITC at desired timepoints, PSB-D cells or BY-2 cells were washed with corresponding medium and observed under a fluorescence microscope (Nikon, Melville, NY, USA) with a filter set Ex488/Em525.…”

Section: Methodsmentioning

confidence: 99%

“…Transcript levels for selected genes were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) as described previously ( Chen et al., 2021 ). Three biological and two technical replicates were performed for each analysis.…”

Section: Methodsmentioning

confidence: 99%

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Differential effects of the recombinant type 1 ribosome-inactivating protein, OsRIP1, on growth of PSB-D and BY-2 cells

Chen

Gistelinck²,

Verbeke³

et al. 2022

Front. Plant Sci.

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Plant suspension cells were treated with recombinant OsRIP1, a type 1 ribosome-inactivating protein (RIP) from rice (Oryza sativa L.). OsRIP1 triggered cell death in tobacco BY-2 cells but not in Arabidopsis PSB-D cells. Phenotypic changes in BY-2 cells exposed to OsRIP1, included loss of growth capacity, loss of integrity of the plasma membrane and vacuolar collapse. These effects were also accompanied by RNA degradation and DNA fragmentation. Targeting of exogenous OsRIP1 to plant vacuoles and OsRIP1-induced accumulation of transcripts for vacuolar processing enzymes (VPEs) indicated that OsRIP1 provoked plant cell death in tobacco BY-2 cells through the activation of VPEs and subsequent vacuolar disruption, which was probably independent of its N-glycosylase activity on cytosolic ribosomes. Necrosis with limited production of H2O2 was observed after infiltration of high concentrations of OsRIP1 in epidermal cells of Nicotiana tabacum cv. Samsun NN plants. Our study provides the first evidence that OsRIP1 exerts differential effects on the growth of PSB-D and BY-2 cells. The vacuole-dependent cell death pathway is associated with the lethal effect of the exogenously applied OsRIP1 on BY-2 cells.

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Section: Discussionmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

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Differential effects of the recombinant type 1 ribosome-inactivating protein, OsRIP1, on growth of PSB-D and BY-2 cells

Chen

Gistelinck²,

Verbeke³

et al. 2022

Front. Plant Sci.

Self Cite

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“…While an indication of the morphology can also be obtained, this is a 2D projection of the 3D real situation. Therefore, other techniques such as SEM, TEM, or AFM are more suited for morphological characterization of cell death modalities 16,54–57 . Since it is unsure what the effect of the suspension medium and the induced shear forces are on the cell death process, the samples were not re‐run through the sDC setup.…”

Section: Discussionmentioning

confidence: 99%

High‐throughput mechano‐cytometry as a method to detect apoptosis, necroptosis, and ferroptosis

et al. 2023

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In recent years, the importance of the investigation of regulated cell death (RCD) has significantly increased and different methods are proposed for the detection of RCD including biochemical as well as fluorescence assays. Researchers have shown that early stages of cell death could be detected by using AFM. Although AFM offers a high single-cell resolution and sensitivity, the throughput (<100 cells/h) limits a broad range of biomedical applications of this technique. Here, a microfluidics-based mechanobiology technique, named shear flow deformability cytometry (sDC), is used to investigate and distinguish dying cells from viable cells purely based on their mechanical properties. Three different RCD modalities (i.e., apoptosis, necroptosis, and ferroptosis) are induced in L929sAhFas cells and analysed using sDC. Using machine learning on the extracted parameters, it was possible to predict the dead or viable state with 92% validation accuracy. A significant decrease in elasticity can be noticed for each of these RCD modalities by analysing the deformation of the dying cells. Analysis of morphological characteristics such as cell size and membrane irregularities also indicated significant differences in the RCD induced cells versus control cells. These results highlight the importance of mechanical properties during RCD and the significance of label-free techniques, such as sDC, which can be used to detect regulated cell death and can be further linked with sorting of live and dead cells.

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“…SEM Analysis: SEM imaging was performed on noncoated and coated ferroptotic MCA cells. Specimen preparation was performed as stated by Chen et al [79] First, the MCA cells were treated with the according cell death inducer (RSL3) and left for 24 h. After performing the LbL coating, the cells were deposited onto poly-L-lysine-coated glasses overnight. Subsequently cells were fixed with 3% glutaraldehyde (Sigma-Aldrich, St. Louis, USA) in PBS buffer for 1 h and post fixed with 1% osmium tetroxide (Sigma-Aldrich) for another hour in the dark at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Mechanobiology of Ferroptotic Cancer Cells as a Novel “Eat‐Me” Signal: Regulating Efferocytosis through Layer‐by‐Layer Coating

Meeren

Efimova

Demuynck

et al. 2023

Adv Healthcare Materials

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The importance of the clearance of dead cells is shown to have a regulatory role for normal tissue homeostasis and for the modulation of immune responses. However, how mechanobiological properties of dead cells affect efferocytosis remains largely unknown. Here, it is reported that the Young's modulus of cancer cells undergoing ferroptosis is reduced. To modulate their Young's modulus a layer‐by‐layer (LbL) nanocoating is developed. Scanning electron and fluorescence microscopy confirm coating efficiency of ferroptotic cells while atomic force microscopy reveals encapsulation of the dead cells increases their Young's modulus dependent on the number of applied LbL layers which increases their efferocytosis by primary macrophages. This work demonstrates the crucial role of mechanobiology of dead cells in regulating their efferocytosis by macrophages which can be exploited for the development of novel therapeutic strategies for diseases where modulation of efferocytosis can be potentially beneficial and for the design of drug delivery systems for cancer therapy.

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The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells

Cited by 5 publications

References 60 publications

Differential effects of the recombinant type 1 ribosome-inactivating protein, OsRIP1, on growth of PSB-D and BY-2 cells

Differential effects of the recombinant type 1 ribosome-inactivating protein, OsRIP1, on growth of PSB-D and BY-2 cells

High‐throughput mechano‐cytometry as a method to detect apoptosis, necroptosis, and ferroptosis

Mechanobiology of Ferroptotic Cancer Cells as a Novel “Eat‐Me” Signal: Regulating Efferocytosis through Layer‐by‐Layer Coating

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