Cellular adaptations to eGFP expression

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2, we sought to investigate the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Wildtype HeLa cells, HeLa cells stably expressing eGFP and HeLa cells expressing eGFP upon doxycycline exposure (tet system for conditional expression) were used. All expreiments were prepared as three independent cell cultures. The cultures of HeLa.tet and HeLa.tet.eGFP at 70% confluency were exposed to 1 ug/mL doxycycline for 48 h before collection. Confluent cells were collected for RNA isolation by trypsinization, washed with PBS and stored at -80°C.