3. MATERIAL AND METHODS
3.1 M ATERIAL
3.1.8 P LASMIDS
Vektor Insert Source Nr.*
pBSK E6AP delta N199 M.Scheffner 98
DHFR DHFR-CYLD wt S.Kühnle 74
MSCV hygro Herc2-1 S.Kühnle 110
MSCV hygro NA8 S.Kühnle 112
MSCV hygro 948i S.Kühnle 114
MSCV neo Herc2-1 S.Kühnle 111
MSCV neo NA8 S.Kühnle 113
MSCV puro 2nd generation NA8 K.Matentzoglu 116
MSCV puro linker Herc2-1 S.Kühnle 109
MSCV puro linker - K.Matentzoglu 122
MSCVNeo NA8 K.Matentzoglu 105
pAlli10 Herc2 3880-ende mit deletion S.Kühnle 26
pBSK Rcc1b (Maus) S.Glockzin 37
pcDNA3-HA Herc2 full-lenght Trenzyme GmbH 36
pcDNA3-HA Ub-LIA (L8A, I44A) S.Kühnle 77
pcDNA3-HA Arc/Arg3.1 K.Matentzoglu 156
pcDNA3-HA UBL (Sacsin) S.Kühnle 158
pcDNA3-HA E6AP P377H o.F.S. S.Kühnle 159
pcDNA3-HA HPV16E7 S.Kühnle 161
pcDNA3-HA ERα S.Kühnle 163
pcDNA3-HA E6AP S349P o.F.S. S.Kühnle 166
pcDNA3-HA Progestrone Receptor S.Kühnle 172
pcDNA3-HA Androgen Receptor (AR) K.Matentzoglu 175
Material 33
pcDNA3-HA Rad23 UBD (XPC) S.Kühnle 177
pcDNA3-HA Rad23DUBL S.Kühnle 178
pcDNA3-HA Rad23 full-lenght S.Glockzin 179
pcDNA3-HA SFPQ K.Matentzoglu 180
pcDNA3-HA Herc2 fragment 14/14 S.Kühnle 1
pcDNA3-HA Herc2 fragment2 S.Kühnle 2
pcDNA3-HA Herc2 fragment D S.Kühnle 3
pcDNA3-HA Herc2 fragment C S.Kühnle 4
pcDNA3-HA Herc2 fragment 2A S.Kühnle 7
pcDNA3-HA Herc2 fragment 2B S.Kühnle 8
pcDNA3-HA Herc2 fragment3 S.Kühnle 9
pcDNA3-HA Herc2 fragment 8-9 S.Kühnle 13
pcDNA3-HA Herc2 fragment10 S.Kühnle 14
pcDNA3-HA Herc2 12618-ende S.Kühnle 17
pcDNA3-HA Herc2 11848-ende S.Kühnle 18
pcDNA3-HA Herc2 11428-ende S.Kühnle 19
pcDNA3-HA Herc2 8152-ende S.Kühnle 23
pcDNA3-HA Herc2 6872-ende S.Kühnle 24
pcDNA3-HA Herc2 6201-ende S.Kühnle 25
pcDNA3-HA empty vektor without bglII restr. site K.Matentzoglu 28
pcDNA3-HA Herc2 fragment 2-3 S.Kühnle 29
pcDNA3-HA Herc2 fragment A+1 S.Kühnle 30
pcDNA3-HA Herc2 wt full-lenght Trenzyme GmbH 38
pcDNA3-HA HA-Rcc1b U.Kogel 39
pcDNA3-HA Herc2 inaktiv full-lenght S.Kühnle 40
pcDNA3-HA HA-Rcc1b-Ende U.Kogel 41
pcDNA3-HA HA-Rcc1b-Ende inaktiv U.Kogel 42
pcDNA3-HA Rcc1 domain of PAM full-lenght S.Kühnle 46
pcDNA3-HA XPA S.Kühnle 49
pcDNA3-HA HA Ring1b I135S (inactive) A.Ciechanover 65
pcDNA3-HA HA Ring1b wt A.Ciechanover 66
pcDNA3-HA HA Ring1b delta RING A.Ciechanover 67
pcDNA3-HA HA-16E6 M.Scheffner 69
pcDNA3-HA HA-CYLD wt K.Matentzoglu 73
pcDNA3-HA Ub-wt S.Kühnle 78
pcDNA3-HA HA-E6AP wt K.Matentzoglu 90
pcDNA3-HA HA-E6AP delta 150-200 S.Kühnle 91
pcDNA3-HA HA-E6AP aa: 150-200 S.Kühnle 92
pcDNA3-HA HA-E6AP Isoform2 K.Matentzoglu 93
pcDNA3-HA HA-E6AP Isoform3 K.Matentzoglu 94
pcDNA3-HA E6AP aa: 420-852 K.Matentzoglu 150
pcDNA3-HA E6AP aa: 491.852 K.Matentzoglu 151
pcDNA3-HA E6AP aa: 1-500 K.Matentzoglu 152
Material 34
pcDNA3-HA E6AP aa: 1-420 K.Matentzoglu 153
pcDNA3.1 GFP-E6AP wt K.Matentzoglu 81
pcDNA3.1 GFP-E6AP delta 150-200 S.Kühnle 82
pcDNA3.1 GFP-E6AP aa:1-420 S.Kühnle 83
pcDNA3.1 GFP-E6AP aa: 420-852 S.Kühnle 84
pcDNA3.1 GFP-E6AP aa:1-500 S.Kühnle 85
pcDNA3.1 GFP-E6AP C820A S.Kühnle 86
pcDNA3.1 GFP-E6AP wt RNAi mut S.Kühnle 87
pcDNA3.1 GFP-E6AP aa:1-420 RNAi mut S.Kühnle 88
pcDNA3.1 GFP-E6AP aa:1-500 RNAi mut S.Kühnle 89
pcDNA3.1 (-) GFP ERb S.Kühnle 155
pcDNA3.1 (-) CFP E6AP act. K.Matentzoglu 160
pcDNA3.1 (-) GFP ERa S.Kühnle 162
pcDNA3.1(-)hygro HA-E6AP delta E6 BS K.Matentzoglu 95 pcDNA3.1(-)hygro HA-E6AP C820A mut NA8 K.Matentzoglu 96
pcDNA3.1(-)hygro HA-E6APC820A K.Matentzoglu 97
pcDNA3.1ha Herc2 fragment U.Kogel 31
pcDNA4TO Herc2-1 S.Kühnle 108
pcDNA4TO 2nd generation NA8 K.Matentzoglu 115
pCMV Myc-PAM K.Matentzoglu 44
pCoc Mdm2 M.Scheffner 143
pet3a UBL-GST(Rad23) S.Kühnle 165
pet3a Ub-LIA (L8A, I44A) H.P.Wollscheid
pet3a Ub-wt H.P.Wollscheid
pFlag -CMV2 Rnf8 wt J.Lucas 59
pgex2tk GST-Rcc1b S.Glockzin 33
pGex2TK GST Rcc1 domain of MYCBP2 S.Kühnle 45
pGex2TK XPA S.Kühnle 50
pGL2 ERE_E1b-Luc C.Smith 167
pGL3basic 3XERE TATA Luc Addgene 154
pRcCMV h-p53 M.Scheffner 142
pRR-PR-5Z PR reporter Addgene 173
pRS NA8 K.Matentzoglu 107
VP16 ERα Addgene 164
* Number plasmid stock 3.1.9 Marker
3.1.9.1 Protein Standards
PageRulerTM Unstained Protein Ladder (Fermentas) PageRulerTMPrestained Protein Ladder (Fermentas)
3.1.9.2 DNA Standards
GeneRuler™ DNA Ladder (Fermentas)
Material 35
3.1.10 Enzymes
3.1.10.1 Polymerases
TAQ-polymerase ,selfmade (AG.Scheffner) Physion-Polymerase (Finnzymes)
3.1.10.2 Phosphatases
Antarctic Phosphatase (New England Biolabs) Alkaline Phosphatase (CIP) (New England Biolabs)
3.1.11 Software
Unicorn5.01 AECTA FPLC device software
AIDA Image Analyzer V4.00 Image editing software, quantification of Western Blots
• Wallac 1420 Manager Wallac1420 device software for
β-galactosidase, Luciferase and Bradford measurements.
• Clone Manager 9 Sequence analysis, oligo-design.
Methods 36
3.2 Methods
3.2.1 Transformation of competent E.coli cells
DNA (5µl ligation sample or 50ng plasmid DNA) was incubated on ice with 50µl of competent E.coli cells (XL-10 Gold, DH5-α)) for 30min. This step was followed by a 90sec heat shock at 42°C. Afterwards cells were incubated on ice for 5min and then transferred either to a flask containing LB medium or to an agar plate containing the selection antibiotic of need. Plates and flasks were incubated at 37°C over night.
3.2.2 Preparation of plasmid DNA
3.2.2.1 Mini Preparation
For mini-preparations 2ml of LB medium containing the respective antibiotic were inoculated with a colony from a LB plate and grown at 37°C over night. The next day the cells were harvested and the plasmid DNA was isolated using alkalyc lysis (Stephen et al., 1990). Therefore the bacteria-pellet was resuspended in 200µl S1 buffer. Cells were then lysed upon addition of S2 buffer for 3min at room temperature.
For precipitation of proteins and genomic DNA, 200µl S2 buffer were added and samples were incubated on ice for 5min. After 30 min of centrifugation at 13.000 rpm, 4°C, the supernantant (600µl) was transferred into a new reaction tube and the plasmid DNA was precipitated upon addition of 400µl isopropanol and centrifugation for 13min 13.000rpm at 4°C. The supernatant was then removed and the pellet was washed with 1ml 70% ethanol upon centrifugation (30min, 13.000rpm) at RT. The ethanol was removed and the pellet was dried in the SpeedVec. The dried pellet was then resolved in 50µl of nuclease-free water and either used for further experiments or stored at -20°C.
3.2.2.2 Midi Preparation
For midi-preparations a 100ml liquid overnight culture was inoculated and incubated in a shaker at 37°C over night. The plasmid DNA was isolated using the PureYield™
Plasmid Midiprep System (Promega) according to the manufacturer’s instructions. The purified plasmids were stored at -20°C.
3.2.3 Determination of DNA concentration
DNA concentration was obtained with the NanoPhotometer (Implen) according to the manufacturer’s instructions.
Methods 37
3.2.4 Restriction digest
Plasmid DNA was digested using buffers and restriction enzymes supplied by NEB (New England Biolabs) according to the manufacture’s instructions. 1-2µg of plasmid DNA was digested in a sample volume of 20-40µl.
3.2.5 Separation of DNA by electrophoresis
Samples were mixed with 10x DNA-loading buffer and separated on 1-2% TAE agarose-gels containing 0.1% ethidiumbromide. The separated DNA fragments were visualized with UV-light at 254nm.
3.2.6 Extraction of DNA from agarose Gels
DNA was isolated from gels using a scalpel and then purified from the gel using the Nuleosoin extraction Kit (Macherey Nagel) regarding to the manufactorer’s instructions.
3.2.7 Ligation of DNA fragments
Ligation was performed using the T4 ligase (Fermentas) in 5x Rapid Ligation Buffer (Fermentas). 10µl ligation reaction was incubated at room temperature for 5-30min, followed by transformation (2.2.1)
3.2.8 DNA sequencing
Sequencing of DNA took place at GATC (Konstanz, Germany) on ABI 3730xl.
3.2.9 Polymerase-chain-reaction (PCR)
Amplification reactions for cloning of DNA fragments were performed with Phusion Polymerase in HF (High-Fidelty) or GC Buffer (Fermentas) according to the manufacture’s instructions. 50µl PCR reaction contained 50ng template DNA, 800000000pM dNTPs, 400nM of forward and reverse oligonucleotides and 1µl of Phusion polymerase. Screening PCRs on Colonies (2.2.10) were performed under similar conditions in a 20µl reaction volume using ThermoPol-Buffer (Fermanetas) and self-made TAQ polymerase.
3.2.10 Preparation of RNA
For RNA isolation, mammalian cells were harvested and the pellet was stored at -80°C till use. Mammalian cell pellets were resuspended in 1ml of TRIzol® Reagent (Invitrogen) and homogenized by vortexing for 30sec, subsequently 200µl of CHCl3
was added, followed by another vortexing step for 30sec. Phase separation was obtained by a centrifugation step of 10min at 11900 rpm at 4°C. The supernatant was transferred to a new reaction tube containing 200µl CHCl2, the samples were again mixed by vortexing for 30s to remove the residual phenol. Afterwards the phases were again separated by centrifugation for 10min at 11900rpm. 350µl of the upper phase were mixed with 350µl 70% EtoH p.A. and applied to a Genejet RNA purification
Methods 38
column (Fermentas). After centrifugation for 1min at 12.000rpm (all following centrifugation steps have been carried out for 1min at this velocity), the flow-through was discarded and the column was washed with 600µl wash buffer 1 (Genejet RNA purification Kit, Fermentas). After centrifugation the flow-through was again discarded and the column was in three steps (1) 200µl, (2) 500µl, (3) 200µl) washed with wash buffer2 (Genejet RNA purification Kit, Fermentas). Afterwards the column was dried by centrifugation and the RNA was eluted by centrifugation in 50µl pre-warmed Nuclease free water. The purified RNA was either directly used for RTreactions or stored at -80°C.
3.2.11 Determination of RNA concentration
RNA concentrations were determined using an Eppendorf Bio- Photometer (Eppendorf) and 70µl micro UV-cuvettes (Plastibrand). RNA was diluted (1:10-1:100) and measured at wavelength 260nm.
3.2.12 Reverse transcription
Reverse Transcription of RNA to cDNA was performed using the SuperScript® III Reverse Transcriptase Kit (Invitrogen). Equal amounts of RNA (2µg) for each sample were incubated with 1µl Oligo dT20 for 8min at 80°C using a master cycler PCR device (Eppendorf). The samples were cooled down to 40°C and further incubated for 10min at 40°C, then a master mix, containing 4µl cDNA reaction buffer, 1µl dNTPs, 1µl DTT, 0,5µl RNAseOUT, 0,5µl SuperScript Reverse Transcriptase and DEPC-treated H20, was added to each sample (20µl sample volume). The RT-PCR was performed under the following conditions: 50°C – 30s, 40°C- 4min30sec, these cycles were repated 8 times, followed by a denaturation step at 90°C for 3min. The samples were then cooled down to 4°C and either stored at -80°C or purified using a GeneJet PCR purification Kit (Fermentas).
3.2.13 Purification of cDNA
The cDNA was mixed with 125µl PB (GeneJet PCR purification kit) and applied to a GeneJet PCR purification Kit column. The column was washed once with 100µl PB, followed by 3 washing steps with 500µl washing buffer (GeneJet PCR purification Kit) and one additional washing step with 200µl washing buffer. The column was then dried by centrifugation and eluted in 50µl elution buffer. CDNA was either stored at -80°C or used for Q-PCR analysis.
3.2.14 Quantitative PCR (Q-PCR)
Maxima™ SYBR Green/ROX qPCR Master Mix was used for Quantitative PCR. The Master Mix was supplemented with specific Oligos and cDNA (1µl) (2.2.13) and the
Methods 39
PCR was performed on the StepOne Quantitative PCR machine (Applichem). For all Q-PCRs the PCR efficiency was determinded using cDNA dilutions for all analyzed targets.
3.2.15 Analysis of Q-PCR data
mRNA expression levels were compared using ΔΔ-Ct method and quantified based on an in-vitro-synthesized absolute standard. Actin mRNA was chosen as reference target. PCR conditions are shown in the table below:
Temperature Duration
Ini. denaturation 95°C 10min
40 cycles Denaturation 95°C 20sec
Annealing 60°C 20sec
Elongation 72°C 1min
Melt Curve
3.2.16 Protein expression in bacteria
Proteins were expressed in E.coli BL21 RIL or E.coli DH5α. Overnight cultures of bacteria were diluted in fresh LB medium to OD600 0.1. Bacteria were then grown to an OD600 of approximately 0.6 and induced with 400µM IPTG (Isopropyl-thio-β-D-galacto- pyranosid; ROTH). After additional 4 hours of growth the cells were harvested by centrifugation. Bacteria pellets were either directly used for protein purification (2.2.17 or 2.2.18) or stored at -80°C.
3.2.17 Affinity purification of GST-fusion proteins
Bacteria pellets (2.2.16) were resuspended in 20ml of PBS 1% Triton and cells were lysed on ice using sonification (3x 20 pulses) (Sonifier W-250 Branson). After sonification the samples were centrifuged at 15.000rpm for 15 min and the supernatant was used for further purification of GST proteins. For that purpose, 100-500µl GSH-beads, equilibrated in PBS 1% Triton, were incubated with the supernatant for 1-4h at 4°C. The beads were then collected by centrifugation and washed for 3 times with PBS 1% Triton. Expression and purification efficiency was determined with SDS PAGE and Coomassie staining. GST proteins were either used for Co-precipitation experiments (Pulldown assays, 3.2.28) or eluted from the beads (3.2.18).
3.2.18 Elution of purified GST fusion proteins
GSH-beads coupled GST proteins were eluted from the beads using GSH-elution buffer (10mM Glutathione in T50, pH7.5). Beads were incubated in elution buffer for 5min on ice. The volume of elution buffer was equal to the volume of beads used for elution.
Methods 40
Elution from beads was repeated for five times and the elution efficiency and protein amount was tested via a Coomassie stained SDS PAGE.
3.2.19 Purification of bacterial expressed ubiquitin
Ubiquitin and the Ubiquitin mutant (LIA) were expressed in BL21 RIL (2.2.16). The obtained bacteria pellets from 1l culture were resuspended in 20ml PBS 1% Triton and lysed by sonification (3x 20 pulses). Afterwards the samples were centrifuged for 15min at 15.000 rpm and the supernatant was used for heat-purification. For that purpose the supernatant was incubated at 70°C for 20min. Then precipitated proteins were removed by another centrifugation step at 20000 rpm for 15min. Due to its heat-stability, ubiquitin remains in the supernatant while most other proteins are precipitated. The supernatant was then aliquoted and stored at -20°C.
3.2.20 SDS PAGE
SDS PAGE was performed according to Laemmli (Laemmli, 1970). The acrylamide concentration of the seperation gel (370mM Tris-HCl, 0.1% SDS, pH 8.8) was adjusted to the molecular weight of the proteins to be analyzed. The Acrylamide concentration of the stacking gel (125mM Tris-Hcl, 0.1% SDS, pH6.8) was always maintained at 5%. Protein samples were mixed with Laemmli-sample buffer and denaturated by boiling for 2-3min. Electrophoretic protein separations were performed at a constant current of 50mA. Proteins were then visualized using either Western Blot with subsequent immunodetection (2.2.25) or Coomassie-Blue staining (2.2.23).
Radiolabeled proteins were visualized using Radiography (2.2.24).
3.2.21 In-vitro ubiquitination assays
For in-vitro ubiquitination assays the ubiquitin-activating enzyme E1 and E6AP were expressed in the baculovirus system. The ubiquitin conjugating enzyme E2 (His-UbcH5b) as well as GST tagged 16E6, Rcc1b and Rcc1c were expressed in E.coli BL21 and purified via the GST or 6xHis tag (2.2.17, 2.2.18). Ubiquitin was either obtained from Sigma or expressed in bacteria (2.2.19). For the ubiquitination reaction 1-2µl
35S-labeled protein was incubated with 50ng E1, 50ng E2 (UbcH5b) and 10µg of ubiquitin, 50ng GST tagged proteins and 50ng E6AP in 40µl reaction volumes.
Additionally each reaction contained 25mM Tris-HCl (pH7.5), 50mM NaCl, 1mM Dithiothreitol, 2mM ATP and 2mM MgCl2. Reactions were incubated at 25°C for a maximum of 2 hours. Afterwards the reactions were electrophorased by SDS PAGE and the 35S labeled proteins were visualized by radiography.
3.2.22 In-vitro thioester assays
For in-vitro thioester assays, the ubiquitin-activating enzyme E1 and E6AP were expressed in the baculovirus system. The ubiquitin-conjugating enzyme E2 (His-UbcH5b) as well as GST-tagged Rcc1b was expressed in E.coli BL21 RIL and purified
Methods 41
via GST or His Tag. Ubiquitin was either obtained from Sigma, expressed in bacteria (2.2.19) or in-vitro translated (2.2.27). For the thioester reaction 50ng E1, 50ng E2 and 50ng E6AP were incubated in 40µl volume reactions with ubiquitin (10µg Ubiquitin (Sigma or bacterially expressed or 5µl in-vitro translated ubiquitin or ubiquitin mutant). Each reaction contained 25mM Tris-HCl (pH 7.5), 50mM NaCl, 0.8mM Dithiothreitol, 2mM ATP and 2mM MgCl2. Reactions were incubated at 25°C for 3-15 min. Afterwards the reactions were electrophoresed with a constant current of 25mA at 4°C. Radio-labeled proteins were visualized by radiography
3.2.23 Coomassie-blue staining
After separation by electophoresis the SDS PAGE was incubated for 1 hour in Coomassie-Blue solution, followed by incubation in destain solution until the desired intensity of staining was reached.
3.2.24 Radiography
To visualize 35S labeled proteins in a SDS Gel, the gel, after seperation by SDS PAGE, was incubated for 1 hour in destain-solution. Afterwards the gel was further incubated in Amplifyer (Amersham) for 1 hour. The gel was then dried under vacuum conditions at 80°C for 1-2 hours and proteins were visualized using an Imaging plate Bas-IIIs (Fuji) and a BAS cassette 2 1040 (Fujifilm). After exposition of 1-24 hours an image was taken with the BAS-1000 (Raytest).
3.2.25 Western blot and immunodetection of proteins
Proteins were transferred from SDS PAGE to PVDF membranes using a blotting chamber (Biorad). The transfer was performed in 1x transferbuffer (12,5mM Tris-HCl, 100mM Glycin, (pH 8.3)). Depending on the size of the transferred protein, the blotting procedure was performed for 90min at a constant current of 60V at room tempereature (small and normal sized proteins) or overnight at a constant current of 30V at 4°C (large proteins > 400kDa)
After the transfer the membranes were incubated in blocking buffer (5% Milk powder (Roth) in TNE-T) either for 1 hour at RT or over night at 4°C.
The blocking buffer was removed and the membranes were excessively washed with TNE-T to remove all residual milk. Afterwards the membranes were incubated with the primary antibody for 1 hour in TNE-T at room temperature or at 4°C over night. After three additional washing steps with TNE-T, the membranes were incubated for 1 hour at room temperature with horse-raddish-peroxidase (HRP) coupled secondary antibody, either anti-rabbit (1:15000 dilution in TNE-T) or anti-mouse (1:20000 dilution in TNE-T). Afterwards the secondary antibody was removed by extensive
Methods 42
washing and proteins were detected with an ECL (enhanced chemiluminescence)-reaction (Perkin Elmer) on the Imaging system Fuji LAS 3000.
3.2.26 Determination of protein concentration
The protein concentration of cell-lysates was determined with BioRad Protein assay (Bradford-reagent) according to the manufacture’s instructions. Measurements were performed at a wavelength of 595nm, lysisbuffer was used as blank value.
3.2.27 In-vitro Translatiion
In-vitro translation of proteins was performed in either reticulocyte-lysate or wheat-germ lysate using the TNTTM coupled reticulocyte lysate (Promega) according to the manufacture’s instructions. For visualization, proteins were radioactive labeled with
35S-Methionine (Perkin Elmer).
3.2.28 In-vitro GST-co-precipitation assays (GST-pulldowns)
The purified and GSH matrix bound GST fusion proteins were equilibrated in TNN buffer and similar protein amounts were added to each pulldown assay. Different bead amounts were adjusted with empty GSH beads. GST coupled to GSH beads were used as negative control. Before use beads were blocked with 5% BSA in TNN for 30min and again washed once with TNN. 10µl of either in-vitro translated protein or recombinant expressed E6AP were added. The volume was adjusted to 300µl with TNN, followed by incubation for a minimum of 4 hours or over night at 4°C. The beads were then washed for 5 times with 1ml of TNN. To elute bound proteins, samples were boiled 5min in 2x Laemmli buffer (2x Laemmli buffer and beads were mixed in the ratio 1:1). Electrophoresis on SDS PAGE was performed and separated proteins were either transferred to a PVDF membrane (Western Blot 2.2.25) or in the case of radioactive samples, the proteins were visualized with radiography (2.2.24).
3.2.29 Cell culture of mammalian cells
Mammalian cells were cultured on CellStar plates (Greiner). Most cell-lines (HEK293T, H1299, RKO, MCF-7) were cultured in DMEM (Gibco) 10%FCS (Gibco) supplemented with 1ml Normocin (Invivogen)/550ml Medium. The neuroblastoma cell line SH-SY5Y was cultured on CellBIND plates (Costar) in DMEM-F12 Glutamaxx (Gibco) 10% Heat inactivated FCS (PAA). NCI-H295R cells were cultured in cell culture dish (Costar) in DMEM-F12 10% Heat-inactivated FCS (PAA). For estrogen-reporter assays cells were cultured in phenol-red free DMEM (Gibco) or DMEM-F12 Glutamaxx (Gibco) supplemented with 5% charcoal stripped FCS (Gibco and see 2.2.15). All cell lines were maintained in an incubator at 37°C 4% CO2.
Methods 43
3.2.30 Transfection of mammalian cells
Cells were transfected with different methods dependent on the cell line and experiment.
3.2.30.1 Lipofection with Lipofectamine 2000
For transfection with Lipofectamine™ 2000 transfection reagent (Invitrogen), cells were seeded on culture plates the day before transfection, the number of cells seeded was chosen in a way that they reached 90-95% confluency on the day of transfection.
Cells were transfected according to the manufacturer’s instructions. 24h after transfection the cells were either harvested or subjected to stable selection.
3.2.30.2 Lipofection with Turbofect
TurboFect™ (Fermentas) transfection was used for „easy-to-transfect” cell lines. The day before transfection, cells were seeded on cell-culture plates to reach 60-80%
confluency on the day of transfection. Transfection was performed according to the manufacturer’s instructions. 24h post transfection, cell were harvested and either directly used for experiments or stored at -80°C.
3.2.30.3 Nucleofection with Amaxa
Cells were detached from the surface by accutase (PAA) treatment, cells were counted using a countess (Invitrogen) and the correct number of cells (1*106) was collected by centrifugation at 800rpm for 10 min at room temperature. The Amaxa™
Nucleofector™ Technology (Lonza AG) was used for transfection of SH-SY5Y cells and NCI-H295R cells. 1*10^6 – 2*10^6 cells were transfected with 2µg of plasmid DNA.
Both cell lines were transfected using Kit V/Supplement1 and the high cell-survival program A-23 on a Nucleofector1 device (Amaxa).
Afterwards cells were seeded on cell-culture plates. 24 hours after transfection the cells were harvested or subjected to stable selection.
3.2.31 Determination of transfection efficiency (ß-gal assay)
To determine the transfection efficiency a ß-galactosidase plasmid DNA was co-transfected in several experiments, the DNA amount of ß-galactosidase displayed 1/10 of the total DNA amount. Transfected cells were lysed in an appropriate volume of
To determine the transfection efficiency a ß-galactosidase plasmid DNA was co-transfected in several experiments, the DNA amount of ß-galactosidase displayed 1/10 of the total DNA amount. Transfected cells were lysed in an appropriate volume of