Browsing by Author "Aslam, Saba"

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    Key cytoskeletal proteins are upregulated in 2D cell culture in comparison to 3D culture
    (2020-01) Aslam, Saba; Ungrin, Mark D.; Krawetz, Roman J.; Nezhad, Amir Sanati
    The cytoskeleton is the structural framework of the cell, and plays an important role in cell motility, signalling pathways, and cell behaviour. It is composed of various proteins that form a dynamic network of filaments and tubules to mechanically support the cell. Commonly, cytoskeletal proteins such as β-actin are used as controls or references in many laboratory techniques because they are assumed to remain stable across various conditions. However, most of the research to date has been performed on 2D monolayers of cells adhered to plastic. Recently, 3D cell culture techniques have become more feasible and increasingly popular as they better represent the physiological microenvironment found in the body. As more research shifts from 2D to 3D culture, the changes in cytoskeleton due to culture conditions need to be better characterized. To study gene expression, eleven human cell lines were cultured in 2D and 3D, and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed. For analysis, ten control genes were evaluated for stability, and the geometric mean of the three most stable genes was used for normalization. The results show a significant downregulation of several cytoskeletal genes such as β-actin, vimentin, and keratin in 3D compared to 2D. Similarly, key cytoskeletal proteins were also found to be downregulated in 3D using Western blots normalized to total protein. These results demonstrate that the microenvironmental differences between culture conditions can considerably influence cytoskeletal component levels across various cell types. Integrating these findings with the published literature, we suspect that the supra-physiological stiff surface in plastic-adherent culture leads to overexpression of cytoskeletal proteins – presumably also impacting many other pathways and cell behaviour. These findings challenge the assumption that β-actin provides a consistently stable reference gene/protein, emphasize the importance of physiologically relevant culture systems, and highlight the need to select appropriate controls for specific experimental conditions.