Fig. 4.

eGFP in cells induces gene expression associated with oxidative stress and other biological processes. Images of HeLa cells and stable eGFP-expressing HeLa cells (HeLa/eGFP) under a fluorescent microscope with a green filter can be seen in (a). eGFP was also detected by Western blotting, with GAPDH staining as the loading control. In (b) extracellular H₂O₂ was determined in HeLa and HeLa/eGFP. Catalase was added to cultures at 500 U/mL. (c) Microarray-based experiments were designed to show the effect of GFP expression in the gene expression of two independent cell models. The two GSEA (Gene Set Enrichment Analyses [20]) were performed using the hallmark dataset (h.all.v5.0.symbols) and 1000 permutations. The contrasts were HeLa/eGFP versus HeLa, and Hela.tet.eGFP + doxycycline subtracted from HeLa.tet.eGFP versus HeLa.tet + doxycycline subtracted from HeLa.tet. The enriched gene sets with p-values lower than 0.001 were ranked, and matches between the two independent analyses are shown highlighted in yellow. The top genes contributing to the enrichment for the commonly enriched gene sets are depicted as heat maps in Supplementary Data Fig. S3. (d) Intracellular levels of HIF1α were determined by Western blotting using whole cell lysates of HeLa cells and HeLa cells stably expressing eGFP. In (e), GAPDH densitometry was used as the loading control to normalize the densitometry of HIF1α. * is used to show statistically significant differences between HeLa and HeLa/eGFP.