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- Bolshakova, Anastayia, et al.
(författare)
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EGF-induced dynamics of NF-kappa B and F-actin in A431 cells spread on fibronectin
- 2015
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Ingår i: Histochemistry and Cell Biology. - : Springer Verlag (Germany). - 0948-6143 .- 1432-119X. ; 144:3, s. 223-235
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Tidskriftsartikel (refereegranskat)abstract
- To evaluate the role of actin cytoskeleton in the regulation of NF-kappa B transcription factor, we analyzed its involvement in the intracellular transport and nuclear translocation of the NF-kappa B RelA/p65 subunit in A431 epithelial cells stimulated with fibronectin and EGF. Live cell imaging and confocal microscopy showed that EGF activated the movement of RelA/p65 in the cytoplasm. Upon cell adhesion to fibronectin, RelA/p65 concentrated onto stress fibers, and EGF stimulated its subsequent allocation to membrane ruffles, newly organized stress fibers, and discrete cytoplasmic actin-rich patches. These patches also contained alpha-actinin-1 and alpha-actinin-4, vinculin, paxillin, alpha-tubulin, and PI3-kinase. Cytochalasin D treatment resulted in RelA/p65 redistribution to actin-containing aggregates, with the number of cells with RelA/p65-containing clusters in the cytoplasm increasing under the effect of EGF. Furthermore, EGF proved to induce RelA/p65 accumulation in the nucleus after cell pretreatment with actin-stabilizing and actin-destabilizing agents, which was accompanied by changes in its DNA-binding activity after either EGF stimulation or cytochalasin D treatment. Thus, EGF treatment of A431 cells results in simultaneous nuclear RelA/p65 translocation and cytoplasmic redistribution, with part of RelA/p65 pool forming a very tight association with actin-rich structures. Apparently, nuclear transport is independent on drug stabilization or destabilization of the actin.
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| 2. |
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| 3. |
- Jobe, Njainday Pulo, et al.
(författare)
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Fibroblasts potentiate melanoma cells in vitro invasiveness induced by UV-irradiated keratinocytes
- 2018
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Ingår i: Histochemistry and Cell Biology. - : Springer Science and Business Media LLC. - 0948-6143 .- 1432-119X. ; 149:5, s. 503-516
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Tidskriftsartikel (refereegranskat)abstract
- Melanoma represents a malignant disease with steadily increasing incidence. UV-irradiation is a recognized key factor in melanoma initiation. Therefore, the efficient prevention of UV tissue damage bears a critical potential for melanoma prevention. In this study, we tested the effect of UV irradiation of normal keratinocytes and their consequent interaction with normal and cancer-associated fibroblasts isolated from melanoma, respectively. Using this model of UV influenced microenvironment, we measured melanoma cell migration in 3-D collagen gels. These interactions were studied using DNA microarray technology, immunofluorescence staining, single cell electrophoresis assay, viability (dead/life) cell detection methods, and migration analysis. We observed that three 10 mJ/cm2 fractions at equal intervals over 72 h applied on keratinocytes lead to a 50% increase (p < 0.05) in in vitro invasion of melanoma cells. The introduction cancer-associated fibroblasts to such model further significantly stimulated melanoma cells in vitro invasiveness to a higher extent than normal fibroblasts. A panel of candidate gene products responsible for facilitation of melanoma cells invasion was defined with emphasis on IL-6, IL-8, and CXCL-1. In conclusion, this study demonstrates a synergistic effect between cancer microenvironment and UV irradiation in melanoma invasiveness under in vitro condition.
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| 4. |
- Libard, Sylwia, et al.
(författare)
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Characteristics of the tissue section that influence the staining outcome in immunohistochemistry
- 2019
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Ingår i: Histochemistry and Cell Biology. - : Springer Science and Business Media LLC. - 0948-6143 .- 1432-119X. ; 151:1, s. 91-96
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Tidskriftsartikel (refereegranskat)abstract
- Immunohistochemistry (IHC) is influenced by several factors such as cold ischemia time, fixative, fixation time, paraffin, storage time, antibody, antigen retrieval technique and detection systems. In the setting of post-mortem tissue, not only post-mortem delay, but also agonal state is of interest. Here, we assessed an additional variable, i.e., the thickness of the section, and noted that this variable also influenced the IHC outcome. This is of significance when the extent of labelling is a parameter to be assessed, for example when assigning a stage or grade of a disease. Furthermore, when assessing brain tissue with neurons, soma measuring from 4 to 100 µm, various cellular compartments composed of different proteins are localised in sections measuring 4 or 7 µm. Thus, what is seen in a 7-µm-thick section might be lacking in a 4-µm-thick section. Lack of information regarding the molecular size of commercial antibodies is also disturbing as this parameter might influence the distribution of the molecule in the three-dimensional section. The choice of antibody to be used and the staining methodology have been acknowledged being of significance for IHC outcome; however, neither sections thickness or the molecular weight has been discussed sufficiently. IHC has been shown to be an unpredictable technique used for assessment of tissue. This emphasises the need for detailed methodological descriptions in publications, the need to acknowledge and to harmonize all eventual pitfalls related to this methodology.
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