Treatment of eukaryotic cells with trichostatin A (TSA) – a broad-spectrum inhibitor of histone deacetylation – is frequently used in studies of epigenetic regulation of gene expression. To be able to investigate the effects of TSA on promoter activity in reporter gene assays it is necessary to secure high transfection efficiency and low toxicity of the transfection reagent. In this study we tested transfection efficiency and toxicity of FuGENE® HD Transfection Reagent in experiments with hepatoma cell line HepG2. We also established a protocol for TSA treatment that guarantees high cell survival and sufficient TSA effect.
Materials and Methods
For this study, HepG2 human hepatocellular carcinoma cells were obtained from DSMZ and cultivated in RPMI supplemented with 10% FCS. For transfection the cells were seeded at 30% confluence in 24-well plates in 1 ml medium per well 24 hours prior to transfection. On the day of transfection FuGENE® HD Transfection Reagent/plasmid complexes were prepared by mixing 100 µl RPMI without serum with desired amounts of the plasmid and FuGENE® HD Transfection Reagent in a 96-well plate. The plate was placed on a shaker for 15 seconds and then incubated at room temperature for 15 minutes. Obtained complexes were then added to the cells.
Trichostatin A was used for the treatment of HepG2 cells at following concentrations: 0.1 µM, 0.25 µM, 0.5 µM,
0.75 µM, 1.0 µM, 1.5 µM.
A Plasmid containing GFP gene pmaxGFP was used in experiments where visualization of transfected cells was needed. For luciferase assays a reporter vector containing synthetic Renilla luciferase gene phRL-TK was used (Promega). Plasmids were isolated using standard methods. The concentration of the plasmid was measured spectrophotometrically and adjusted to 1 µg/µl.
Results and Discussion
Initial optimization of transfection protocol for HepG2 cells was performed according to the standard scheme using pmaxGFP plasmid. Transfection was performed in 24-well plates in a final volume of 1 ml. For the preparation of transfection complexes, 1 µg or 2 µg of the plasmid were mixed with 3 µl, 4 µl, 5 µl, 6 µl, 7 µl, 8 µl, 9 µl, or 10 µl of FuGENE® HD Transfection Reagent for each well. Transfection efficiency was determined by counting the number of GFP-positive cells. Highest transfection efficiency (about 70% of transfected cells) was obtained when the transfection mix contained 1 µg of the plasmid and 3 µl of FuGENE® HD Transfection Reagent. No significant difference in cell viability in different wells was observed.
Next the concentration at which TSA is toxic for HepG2 cell was determined. The cells were cultivated for 72 hours in the presence of TSA used at concentrations indicated in materials and methods. It was found that only the concentration of 1.5 µM was toxic for the cells. Therefore, for the combined transfection/TSA treatment experiment TSA was used at a concentration of 0.5 µM. Culture medium was supplemented with TSA 24 hours prior to transfection. Transfection was also performed in the presence of 0.5 µM TSA. We found that combining continuous TSA treatment with FuGENE® HD Transfection Reagent leads to 100% lethality of treated cells as early as 24 hours after transfection. To identify conditions that would allow inhibition of histone deacetylases in transfected cells we analyzed the influence of TSA treatment timing on HepG2 cells vitality (Figure 1a). We found that TSA used at a concentration of 0.5 µM was not toxic for the cells only if the treatment was performed during the last 24 hours of transfection (Figure 1a, lane 2). Some vital and transfected cells were also observed if TSA treatment was performed only 24 hours prior to transfection (Figure 1a, lane 1), but the number of these cells was too small to be considered as acceptable for routine experiments.
Using the established protocol, we analyzed the effect of TSA treatment on the expression of Renilla luciferase gene in cells transfected with phRL-TK plasmid (Figure 1b). Transfection was performed using 1 µg plasmid and 3 µl FuGENE® HD Transfection Reagent and Renilla luciferase activity was measured. We found that inhibition of histone deacetylases resulted in dramatic increase of Renilla luciferase expression.
The established protocol allows to use FuGENE® HD Transfection Reagent for the analysis of histone deacetylases in the regulation of gene expression in HepG2 cells. The optimization scheme used by us will allow easy adaptation of the transfection/TSA treatment protocol for other cells that are difficult to transfect.
This article was originally published in Biochemica 2/2009, pages 6-7. ©Springer Medizin Verlag 2009