Cell biological methods

2.16.1 Cell culture of eukaryotic cells

Cells were cultured in their appropriate medium (see section 2.8.3) in surface-treated cell culture flasks (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator with 5 % CO2. Depending on the proliferation rate and confluency of the cells, they were splitted once or twice per week. For the splitting process the cells were first washed with DBPS (PAN Biotech GmbH, Aidenbach, Germany) and then incubated for a few minutes at 37 °C in a minimal amount of Trypsin/EDTA (PAN Biotech GmbH, Aidenbach, Germany) for detachment from the culture flask.

Using an inverted microscope it was controlled whether all cells were detached. If this was the case, the trypsin reaction was stopped by adding growth medium. Depending on proliferation rate of the cells they were diluted either 1:5 or 1:10 with fresh culture medium.

2.16.2 Cryo-preservation and revitalization of eukaryotic cells

For cryo-preservation, the cells were grown to 80 % confluency and then trypsinized as described in 2.16.1. When all the cells had detached from the bottom of the cell culture flask the

tryspin reaction was stopped with medium. The cells were transferred to a tube and centrifuged at 200 x g for 5 min to form a cell pellet and remove the remaining trypsin. The pellet was resuspended in the appropriate amount of culture medium and then diluted 1:1 with cryo-medium (see 2.8.3). Overnight, the cells were kept in Mr. Frosty (Thermo Scientific, Langenselbold, Germany) in a -80°C freezer where the cells were slowly cooled (1°C/min). The following day, the cells were transferred to liquid nitrogen for long-term storage. When cells were revitalized, they were quickly thawed and transferred to a tube containing 5 ml of the appropriate medium.

The cells were centrifuged at 200 x g for 5 min, the supernatant was removed and the cell pellet was resuspended in pre-warmed culture medium. The cells were then transferred to a cell culture flask and kept in the incubator. The next day the medium had to be changed in order to fully remove remaining DMSO.

2.16.3 Test for Mycoplasma contamination

To detect a possible contamination by Mycoplasma, a routinely test was conducted every month.

Therefore, the MycoAlert® Mycoplasma Detection Kit (Lonza, Cologne, Germany) was used according to the manufacturer’s instructions, except that only half of the recommended amounts were used. The principle of this test is the measurement of the activity of mycoplasma-specific enzymes before and after application of specific substrates.

2.16.4 Transfection of eukaryotic cells

2.16.4.1 Transfection of plasmids into eukaryotic cells

Eukaryotic cells were transfected with plasmids in order to induce an overexpression of fusion proteins (pIRES2-EGFP-CCND2, pEBTetD-CCND2) or to induce a downregulation of the fusion protein by shRNA (pSingle-tTs-Ccnd2-shRNA). Two different transfection reagents were used:

Lipofectamine® Transfection Reagent (Thermo Fisher Scientific, Langenselbold, Germany) or Metafectene® Pro (Biontex Laboratories GmbH, Munich, Germany). Both transfection reagents contain lipids which form vesicles with a bilayer sheet, so called liposomes, in aqueous solution.

The liposomes can form nucleic acid lipid complexes with nucleic acids. These complexes can actively be taken up by the eukaryotic cells by a process called endocytosis. The nucleic acid can enter the nucleus only if the nuclear membrane dissolves during mitosis. Therefore, the division rate of cells is critical for DNA transfection and must be as high as possible for efficient transfection. The cells were plated in a sufficient number into 6-well cell culture plates or T-25

cell culture flasks so that the following day a confluency of 70 – 90 % was reached. For the transfection, plasmid DNA was mixed with the transfection reagent according to the manufacturer’s instructions. The mix was incubated for 20 – 30 min and then applied dropwise to the cells in normal growth medium, which were prior washed with DPBS. The cells were incubated overnight at normal culture conditions. The following day, the transfection reagent- containing medium was replaced by fresh medium.

2.16.4.1.1 Generation of single-cell clones/populations

Cells transfected with the pEBTetD-CCND2 plasmid were treated from the next day on with puromycin for clonal selection of successfully transfected cells (see Chapter 2.8.3.). The surviving cells were further cultured until the amount of cells was sufficient to test by western blot analysis if the cells indeed exhibit a cyclin D2 overexpression. Total protein was isolated after the cells were treated with doxycycline in different concentrations (0.01 µg/ml – 1 µg/ml) for 48 or 72 hours to induce cyclin D2 overexpression. Successfully generated cells with a doxycycline-inducible cyclin D2 overexpression were preserved by cryo-preservation and subject to proliferation studies.

Cells transfected with the plasmid pIRES2-EGFP-CCND2 or pSingle-tTs-Ccnd2-shRNA underwent a serial dilution with normal growth medium, ranging from 1:10 – 1:80, in order to generate single-cell clones. This was done two days after the transfection. The diluted cells were plated in cell culture petri dishes and cultured under normal cell culture conditions. From the next day on the cells received Geneticin disulfate (G418)-solution with their normal growth medium in order to select positively transfected cells. Approximately three weeks after the serial dilution of the cells, single cells had formed clones which were picked with a pipette, transferred to a 96-well plate and incubated at normal culture conditions. When a confluency of 70 – 90 % was reached the cells were transferred to the next bigger well until again a confluence of 70 – 90 % was reached and so on (96-well  24-well  12-well 6-well  T-25 flask), until finally the amount of cells was sufficient to fill a T-75 cell culture flask. Total protein was isolated from the cells and via western blot analysis the overexpression or downregulation of cyclin D2 within the transfected cells was tested. Cells transfected with pSingle-tTs-Ccnd2-shRNA had to be treated with doxycycline for 48 – 72 hours prior to protein isolation in order to induce transcription of the shRNA. Cells that were tested positive for cyclin D2 overexpression or cyclin D2 downregulation were conserved by cryo-preservation and were subjected to proliferation, migration or soft agar assays.

2.16.4.2 Transfection of small interfering RNA (siRNA) into eukaryotic cells

siRNAs were used for silencing of a specific gene. siRNAs bind to a specific mRNA, thereby marking it for degradation which results in a reduced gene expression. Three different mouse siRNAs against cyclin D2 (MSS236126, MSS236127, MSS236128, Stealth RNAi™ siRNA, Life Technologies, Darmstadt) and three different human siRNAs against cyclin D2 were used (HSS101452, HSS101453, HSS101454, Stealth RNAi™ siRNA, Life Technologies, Darmstadt).

Human and mouse PCa cells were transfected with gene-specific siRNAs by using the siRNA transfection reagent Oligofectamine™ (Life Technologies, Darmstadt, Germany). Thereby complexes of lipids and siRNA oligonucleotides will be formed which facilitate the uptake of the siRNA molecule into mammalian cells. A sufficient amount of the cells were plated into a 6-well plate or T-25 culture flask so that the following day for transfection a confluence of 50 % was reached. The transfection reaction was pipetted according to the manufacturer’s instructions with a final concentration of 50 nM siRNA- duplexes. Control PCa cells were transfected with siRNA- duplexes against the luciferase gene Photinus pyralis. The transfection reaction was incubated for 30 min at RT in order to form the lipid-siRNA-complexes. In the meantime the cells were washed with DBPS (PAN, Aidenbach, Germany). OptiMEM I (Life Technologies, Darmstadt, Germany) was added to the cells as well the transfection reagent, which was applied dropwise.

The cells were cultured overnight at normal culture conditions in the 37°C incubator. The following day the transfection reagent-containing medium was replaced by normal growth medium. Either protein or RNA was isolated from the transfected cells at different time points or the cells were further subjected to migration or soft agar assays.