General cell culture methods

Working with cells was always performed in a laminar flow hood under sterile conditions. Equipment, material and solutions were sterilized and equipment and material was additionally disinfected by wiping with 80% ethanol prior to usage under the laminar flow hood. Mycoplasma testing and cell authentication was conducted for every newly acquired cell line. Additionally, mycoplasma and cell authenticity testing were conducted regularly. For this purpose, either an analytical PCR to detect Mycoplasma specific genes was performed using the AmpliTaq Gold® DNA polymerase (see 2.2.1.6) or a cell pellet was sent to Multiplexion (Multiplexion, Immenstaad) for a comprehensive cell contamination test or cell authentication test.

2.2.3.1 Freezing and thawing of cells

DMSO was used as a cryoprotective agent hindering crystallization of the cells. For the freezing procedure, cells were grown to a confluency of 90% in a T75 cell culture flask. First, cells were trypsinized for approximately 2min, harvested by centrifugation (3min, 400xg) and the supernatant was discarded. Cells were counted and 1x106-1x107 cells were resuspended in 1ml of the corresponding culture medium. Then, 1ml of freezing medium was added to reach a final DMSO concentration of 10%, mixed gently and the suspension was transferred to a 2ml cryogenic vial. This step had to be performed quickly, because of the cytotoxicity of DMSO. The tubes were immediately placed into a pre-cooled (4°C) Cryo-Safe cryogenic vial cooler allowing a gradual temperature drop of

MATERIALS & METHODS

1°C/min, and kept in a -80°C freezer. On the next day, the tubes could be placed into the liquid nitrogen tank for permanent storage.

Also the thawing procedure had to be performed quickly, because of the cytotoxicity of DMSO. 20ml of pre-warmed culture medium was filled into 50ml falcons. Cells were rapidly thawed in a 37°C water bath and then transferred to the prepared falcon and distributed properly. Subsequently, they were centrifuged (3min, 400xg) and the supernatant was decanted. Cells were washed once in their respective cell culture media and then they were put into in a suitable cell culture flask or cell culture plate depending on the cell number and the particular cell type. The following day, the culture medium was exchanged to remove any residual DMSO.

2.2.3.2 Culturing and passaging of cells

In this study, all cell lines were cultured in an incubator at 37°C, 95% relative humidity and 5% CO2 in

either cell culture flasks or cell culture plates. While adherent cells were grown in horizontally lying flasks, suspension cells were grown in flasks standing upright. The flasks or well plates were always replaced after each second cell passage. Cells were split when they reached a confluency of 80-100%.

Adherent cells were passaged by removing the medium and gentle washing with 1xPBS to remove residual medium which could interfere with the subsequent trypsinization. Then, cells were detached by incubation with a suitable volume of trypsin-EDTA solution completely covering the cell layer at 37°C (e.g. 2ml for a T75 cell culture flask). Cell detachment was checked by microscopy. When cells were detached, the reaction was stopped by the addition of serum-containing medium (at least five times the volume of the trypsin solution) and pipetting up and down. Cells were transferred to a 50ml falcon and centrifuged (3min, 400xg). After removal of the supernatant, cells were resuspended in an appropriate amount of fresh culture medium (e.g. 10ml medium in a T75 cell culture flask) and either split at ratios depending on the particular cell type or used for experiments.

Suspension cells were harvested from the flask or plate, centrifuged (3min, 400xg) and the supernatant was discarded. Then, they were resuspended in fresh culture medium and for splitting a desired amount of cells was added to a fresh culture plate or flask. Approximately, 0.5-1x106cells/ml were seeded depending on the growth characteristics of the particular cell type.

2.2.3.3 Counting of cells

Cells were harvested via trypsinization (see 2.2.3.2), diluted in 1xPBS and then mixed 1:1 with Trypan blue stain for life-dead discrimination. Cells were either counted employing a Neubauer counting

MATERIALS & METHODS

cell number/ml =_{number of counted large squares x dilution factor x chamber factor (10000)}counted cells in large squares

2.2.3.4 Treatment of cells with IFNγ

In this study, cells were treated with recombinant human IFNγ to analyze the expression of APM components under these conditions either by qRT-PCR, Western blot or flow cytometry. Cells were grown until they reached a confluency of 70-80%. Then, medium was removed and exchanged for fresh medium. IFNγ was diluted in 1xPBS and added with a final concentration of 50ng/ml. Cells were cultured for another 48h.

The IFNγ used within this study had a specific activity of 5x107IU/mg protein and reached an antiviral bioassay titer of 5.5x107IU determined in the lab of Prof. Rainer Zawatzky (Prof. Frank Rösl, Viral Transformation Mechanisms, DKFZ, Heidelberg).

2.2.3.5 Transfection of eukaryotic cells with siRNA

Knockdown of ERAP1

ERAP1 (NCBI ID: 51752) was knocked down with a siPOOL from siTOOLs BIOTECH. As a

negative control a non-targeting siPOOL was used. The experiments were either conducted in 12-well plates or in 10cm ø dishes for subsequent IPs (see 2.2.2.6). First, cells were trypsinized (see 2.2.3.2), counted (see 2.2.3.3) and diluted in 900µl (for one well of a 12-well plate) of the respective culture medium. Depending on the cell size and growth properties different amounts of cells were needed for the knockdown experiments:

Table 2.9: Cell numbers and siRNA concentration needed to efficiently knockdown ERAP1.

Cell line Cell number per well siRNA concentration

CaSki 1x105 10nM 866 5x104 5nM SNU17 4x105 5nM SNU703 3x105 8nM SiHa 1x105 10nM FK16A 1x105 10nM

The siRNA dilution was prepared in RNase-free water provided by the company. The siRNA pool was in a volume of 10µl and 40µl of Opti-MEM® was added. For one reaction 2µl of the transfection reagent RNAiMAX was diluted in 48µl of Opti-MEM®. The siRNA mix and the transfection reagent mix were combined (1:1) and incubated for 5min at room temperature. Afterwards, 100µl of the mix was transferred per well of a 12-well plate and the cell suspension was added. For 10cm ø dishes, cell number, medium and siRNA/RNAiMAX were scaled up proportionally. Cells were incubated for 72h if not indicated otherwise.

MATERIALS & METHODS

Knockdown of HPV16 E6 and E7

Knockdown of the HPV16 oncogenes E6 and E7 was performed in cooperation with Prof. Karin Hoppe-Seyler (Prof. Felix Hoppe-Seyler, Molecular Therapy of Virus-Associated Cancers, DKFZ, Heidelberg). HPV16 E6 and E7 were knocked down using three specific siRNAs (see 2.1.12) that target all three HPV16 E6 and E7 transcript classes. As a negative control a non-targeting siRNA was used that had at least 4 mismatches to all known human genes. CaSki cells were transfected using DharmaFECT I based on the manufacturer’s instructions. All three siRNAs were pooled at equimolar concentrations to reach a final siRNA concentration of 10nM. Cells were incubated with siRNAs for 72h. Afterwards, cells were lysed (see 2.2.2.2) and used for semidry Western blot analysis (see 2.2.2.4).