Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.
In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.
It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.
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