Immunofluorescence Microscopy

To optimize cell growth on coverslips and the quality of immunofluorescence microscopy, the glass slides were treated before usage. Therefore, the glass was washed for 30 min in an acetone/EtOH solution (1:1) on a horizontal shaker. Thereafter, they were thoroughly rinsed in MilliQ water and dipped in PBS. Excessive PBS (drop) was removed on a paper towel and the coverslip was air-dried.

Directly before usage, the coverslips were microwaved 2x 2 min for sterilization purposes.

Detection of CPDs:

2.5x10⁵ HeLa S3 were seeded on pre-treated coverslips in 12-well plates and incubated overnight. 30 min prior to the experiment medium was exchanged to DMEM with or without 1 µM ABT888. For UV-C irradiation, medium was removed and the cells washed once in prewarmed PBS. PBS was removed again and the cells were irradiated with an UV germical lamp at a wavelength of 254 nm with a dose of 5 J/m². After irradiation, prewarmed medium without PARP-1 inhibitor was added for the indicated time periods. Cells were fixed with 4 % PFA/PBS for 15 min at RT and washed in PBS-T thrice shortly and twice for 10 min. To denature the DNA cells were incubated in 70 mM NaOH/PBS for 8 min. Cells were washed again as above and incubated in 20 % FCS/PBS for 1 h at RT. First antibody incubation was performed overnight at 4°C with an anti-CPD antibody (1:1,000 in 5 % FCS/PBS) in a wet chamber. On the next day, the coverslips were washed as above and blocked with 20 % FCS/PBS twice shortly and twice for 10 min. Secondary antibody incubation was performed for 1 h at RT in the dark (1:400 goat-anti-mouse Alexa Fluor 488 in 5 % FCS/PBS). Again, the coverslips were washed with PBS-T thrice shortly and twice for 10 min. Finally, nucleus counterstaining was performed for 10 min at RT (200 ng/ml), cells were washed 3x 3 min in PBS and embedded in Aqua-Poly/Mount.

Pictures of at least 100 cells were taken with an Axiovert 200M microscope (40x). Mean fluorescence intensity was determined using ImageJ.

Detection of BPDE-DNA:

1x10⁵ HeLa Kyoto cells were seeded on pre-treated coverslips (15 mm) in 12-well plates and incubated overnight. On the next day, medium was removed and incomplete DMEM (w/o supplements) with BPDE (1 and 20 µM) or solvent (0.2 % of total volume) was added for 1 h at 37°C. Afterwards cells were washed three times for five minutes in PBS. Then fixing was performed with methanol:acetone (1:1) for 20 min at -20°C and the cells were allowed to air dry. In a next step cells were treated with 0.05 M HCl for 5 min on ice. After three 5 min washing steps in PBS, a first antigen retrieval was performed by treatment of cells with RNaseA (100 µg/ml in 150 mM NaCl, 1 mM sodium citrate) for 1 h at 37°C. For 3 min the cells were washed subsequently in PBS, 35 %, 50 %, 75 % EtOH. Next, DNA was denatured in 150 mM NaOH in 70 % EtOH for 4 min, then washed twice shortly in PBS.

Cells were incubated for 2 min each in 70 % EtOH containing 4 % formaldehyde (w/v), in 50 % and 35 % EtOH. DNA was stained using Hoechst33342 (200 ng/ml in PBS) for 10 min at RT. Another

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antigen retrieval step was performed with ProteinaseK (10 µg/ml in 20 mM Tris, 1 mM EDTA, pH 7.4) for 10 min at 37°C. Cells were washed again three times 5 min in PBS before unspecific antibody binding was blocked by incubation in PBS/20 % FCS for 1 h at RT. Cells were washed thrice in PBS/0.05 % Tween 20 for 5 min and incubated upside-down in 1^st antibody against BPDE-DNA adducts (1:50 in PBS/5 % FCS) for 1 h at 37°C in a humid chamber. Cells were washed thrice again in PBS/0.05 % Tween 20 and then incubated upside-down in 2^nd antibody (1:400 GαM-Alexa Fluor 488 in PBS/5 % FCS) for 1 h at 37°C in a humid chamber. The coverslips were washed three times 5 min in PBS before they were mounted with Aqua-Poly/Mount on the slides.

Pictures of at least 100 cells were taken with an Axiovert 200M microscope (40x). Mean fluorescence intensity was determined using ImageJ.

Detection of γH2AX and EdU:

1x10⁵ HeLa Kyoto and HeLa Kyoto PARP-1 KO1 cells were seeded on coverslips in 12-well plates one day before the experiment. 20 min before treatment cells were incubated in 10 µM EdU/DMSO, which was also present during BPDE exposure (1 h, 150 nM). To completely remove BPDE and EdU, cells were washed thrice with PBS, and subsequently incubated in full growth medium. 1, 2, 4 and 8 hours after damage induction cells were fixed with 4 % PFA/PBS for 20 min at RT. After a 5 min washing step with PBS, 50 mM NH4Cl/PBS was added for 10 min. Cells were washed again twice for 5 min with PBS, permeabilized with 0.2 % Trition X-100/PBS for 4 min and washed again (2 x 5 min in PBS).

Blocking occurred in 1 % BSA/PBS for 30 min at RT before cells were incubated overnight at 4°C in primary antibody (mouse anti-γH2AX, 1:500 in blocking buffer) upside-down in a humid chamber. On the next day, cells were washed in three steps for 5, 10 and 15 min in PBS. Secondary antibody (goat-anti-mouse Alexa Fluor 488) was diluted 1:400 in blocking buffer and for 45 min at RT cells were incubated therein upside-down in a humid chamber. After that, cells were washed again for 5, 10 and 15 min with PBS.

To detect the incorporated thymidine analogue EdU a click-reaction was performed with a fluorescent azide (Click-iT Plus Kit). Therefore, the reaction buffer was prepared in the order listed in Table 3.21.

1x Reaction Buffer and 1x Reaction Buffer Additive were diluted freshly in MilliQ water.

Table 3.21: Click-iT Plus reaction cocktail.

Reaction Component Volume

for 24 coverslips

1x Reaction Buffer 830 µl

CuSO4 40 µl

Alexa Fluor Azide647 2.4 µl

1x Reaction Buffer Additive 100 µl

Cells were incubated for 30 min with the Click iT Plus reaction mix at RT in a humid chamber upside-down. Slides were washed for 5 min with PBS before nuclear counterstaining with Hoechst33342 (200 ng/ml) for 15 min at RT was conducted. Finally, cells were washed again thrice in PBS and then embedded in Aqua-Poly/Mount.

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Confocal microscopy (LSM780) was performed and images taken comprising more than 100 cells of each, EdU positive and EdU negative cells, per experimental setting. Data were evaluated using ImageJ and numbers of γH2AX foci per cell were defined using the BIC Macro Toolkit.

Detection of γH2AX and 53BP1:

Sample treatment and preparation was performed as described for the γH2AX/EdU staining with some variations. Here, an EdU staining was not performed, thus cells were neither incubated with EdU nor was the click-reaction performed. Instead, a co-staining for 53BP1 was performed. Therefore, simultaneous to the 1^st AB staining with γH2AX, cells were co-incubated with rabbit anti-53BP1 antibody (1:200). The same applies for the incubation with the secondary antibody (goat-anti-rabbit Alexa Fluor 568, 1:400).

Confocal microscopy (LSM780) was performed and images were taken with more than 100 cells per experimental setting. Data were evaluated using ImageJ and numbers of colocalizing γH2AX and 53BP1 foci per cell were defined using the BIC Macro Toolkit.

Detection of XPA-eGFP:

See ‘Analysis of PAR Formation after Laser-Induced DNA Damage’.

Detection of PARP-1:

1x10⁵ U2OS cells were seeded in µ-dishes 1 day before transfection with XPA-GFP expression plasmid (pEGFP-N1::XPA-eGFP). DNA damage was laser induced as described in 3.2.5.13.1. Approximately 4 min after damage induction cells were fixed in 4 % PFA/PBS for 20 min at RT. µ-dishes were washed shortly with PBS and incubated for 1 min in 100 mM glycine at RT to inactivate remaining PFA. Cells were washed twice in PBS for 5 min and permeabilized in 0.5 % Triton X-100/PBS, washed twice in PBS again and incubated in blocking solution (1 % BSA in PBS) for 1 hour at RT. PARP-1 was stained by incubation of the cells in first antibody FI23 (1:300 in PBS-MT) for 1 h at 37°C in a humid chamber.

After three 5 min washing steps, cells were incubated in secondary antibody (1:400 goat-anti-mouse Alexa Fluor 546 in PBS-MT) for 1 hour at 37°C in a dark humid chamber. After washing 3x 10 min in PBS, nucleus was counterstained with Hoechst33342 (200 ng/ml in PBS) for 5 min. Cells were washed again three times in PBS (5 min) and kept in PBS at 4°C in the dark and analysed near-term.

An Axiovert 200M microscope (40x) was used to take exemplary pictures of PARP-1 recruitment to sites laser-induced DNA damage.

Detection of PAR:

Analysis of ABT888 Stability over Time:

1x10⁵ HeLa S3 cells were seeded on coverslips in 12-well plates the day before the experiment. In case of PARP-1 inhibition, medium containing 1 µM ABT888 or 5 µM PJ-34 were added. 30 min and 8 hour after inhibitor treatment, PAR formation was induced with 0.5 mM H2O2 (in PBS/1 mM MgCl2) for 5 min. Cells were washed shortly in PBS/1 mM MgCl2 and fixed with an ice-cold methanol/acetate solution (3:1) for 7 min at -20°C. Cells were washed 3x 5 min in PBS and unspecific antibody binding was blocked for 1 h in PBS-MT at 37°C.

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Analysis of Effectiveness of PARP Inhibition after ABT888 Removal:

1x10⁵ HeLa Kyoto cells were seeded on coverslips in 12-well plates the day before the experiment. In case of PARP-1 inhibition, medium containing 10 µM ABT888 was added for 30 min. According to the BPDE treatment, PARP inhibition with

ABT888 was performed in incomplete medium (w/o supplements). For untreated control only medium was changed. After the incubation time the inhibitor was replaced with fresh medium. 0 min, 30 min and 1 hour after ABT888 treatment, 0.8 mM H2O2 (in PBS/1 mM MgCl2) was added for 5 min.

Cells were washed shortly in PBS/1 mM MgCl2 and fixed with an ice-cold methanol/acetate solution (3:1) for 7 min at -20°C. Cells were washed 3x 5 min in PBS and unspecific antibody binding was blocked for 1 h in PBS-MT at 37°C.

Analysis of Time Course of PAR Formation (H2O2):

1.5x10⁵ HeLa Kyoto cells were seeded on coverslips in 12-well plates the day before the experiment.

PAR formation was induced with 0.5 mM H2O2 (in PBS/1 mM MgCl2) for the indicated time periods.

Cells were washed shortly in cold PBS/1 mM MgCl2 and fixed with an ice-cold methanol/acetate solution (3:1) for 7 min at -20°C. Cells were washed 3x 5 min in PBS and unspecific antibody binding was blocked for 1 h in PBS-MT at 37°C.

Analysis of PAR Formation after Laser-Induced DNA Damage:

1x10⁵ U2OS cells were seeded in 35 mm µ-dishes one day before transfection with the XPA-GFP expression plasmid (pEGFP-N1::XPA-eGFP). DNA damage was laser-induced as described in chapter 3.2.5.13.1. Since a co-detection of XPA-eGFP was desired, in this setup the classic fixative for PAR detection (MeOH/acetate) was switched to PFA to not quench the eGFP signal. Approximately 1 min after damage induction cells were fixed in 4 % PFA/PBS for 20 min at RT. µ-dishes were washed shortly with PBS and incubated for 10 min in 50 mM NH4Cl/PBS at RT to inactivate remaining PFA.

Cells were washed twice in PBS for 5 min and permeabilized in 0.5 % Triton X-100/PBS, washed twice in PBS again and incubated in blocking solution (1 % BSA in PBS) for 30 min at RT.

PAR was stained by incubation of the cells in first antibody 10H (1:300 in PBS-MT) overnight at 4°C in a humid chamber (1 hour at 37°C for PAR formation after laser irradiation). After three 5 min washing steps cells were incubated in secondary antibody (1:400 goat-anti-mouse Alexa Fluor 488 or Alexa Fluor 546 in PBS-MT) for 1 hour at 37°C in a dark humid chamber. After washing 3x 10 min in PBS, nucleus was counterstained with Hoechst33342 (200 ng/ml in PBS) for 5 min. Cells were washed again three times in PBS (5 min) before embedding the coverslips in Aqua-Poly/Mount. Cells in µ-dishes were not embedded, but kept in PBS at 4°C in the dark and analysed near-term.

Pictures of at least 100 cells were taken with an Axiovert 200M microscope (40x), besides for cells in µ-dishes for which only exemplary pictures were taken. Mean fluorescence intensity was determined using ImageJ.

Figure 3.1: Scheme of damage induction. HeLa Kyoto cells were treated for 30 min (red line) with 10 µM ABT888.

Subsequently, the inhibitor was removed and cells were incubated for the indicated times (blue line). At t0 cells were incubated for 5 min in H2O2 and after 5 min cells were fixed and prepared for immunofluorescence based PAR detection.

- 77 - 3.2.5.14 Flow Cytometer Based Analysis

3.2.5.14.1Host Cell Reactivation Assay (HCRA) UV-Irradiated Reporter Plasmid:

The Host Cell Reactivation Assay (HCRA) is a method to investigate the ability of a cell to repair damaged DNA of exogenous origin. Functional restoration of a UV-C irradiated plasmid was measured to quantify the PAR-dependent NER capacity of UV-DNA lesions and was performed as described earlier ⁵⁴⁶.

Human foreskin fibroblasts were treated with none, 1 or 10 µM ABT888 for 30 min. Thereafter, cells were harvested with trypsin and the resulting cell suspension was divided in two equal aliquots. One aliquot was transfected with a plasmid mix consisting of 3 µg of pEGFP and 15 µg pDsRed plasmid (preparation 1). The other aliquot was transfected with a mix of the same plasmid quantities, but here the pDsRed plasmid had been previously irradiated with 5 kJ/m² UV-C light using a 1800 Stratalinker UV Crosslinker (preparation 2). Transfection was performed in a 4 mm gap cuvette at 0.32 kV and 500 µF with a GenePulser II. After electroporation cells were seeded in 6-well plates in the presence or absence of the respective ABT888 concentrations. 24 hours later cells were harvested with trypsin and the cellular fluorescence signals were analysed with a FACSCalibur flow cytometer. Repair capacity was calculated from relative amounts of red fluorescent cells compared to green fluorescent cells between preparation 1 and 2 for each treatment. The data were normalized to the untreated control.

Z [\]^ B\[\B]_` % = Zb2 + eb2

fb2 + eb2 ∗ fb1 + eb1

Zb1 + eb1 ∗ 100

Zb = ^ g hijk^ lB B B iil fb = m^ hijk^ lB B B iil

eb = gkjni [kl]_]o B iil m^ \ g ^ g hijk^ lB B

pj n ^l ] g]B\_ _kq\^gl [^ [\^\_]k 1 k_ ]^^\g]\_ g k^ [^ [\^\_]k 2 ]^^\g]\_ g .

BPDE-Treated Reporter Plasmid:

To analyse the PAR-dependent NER capacity of BPDE-DNA lesions a HCRA was performed as described above. But instead of UV-C irradiation the pDsRed plasmid was treated with BPDE to induce bulky DNA adducts. 500 µg/ml pDsRed were treated with 0 and 75 µM BPDE in the dark. After a 3 hour incubation time the plasmids were frozen at -20°C and stored until further processing as described before.

3.2.5.14.2Cell Cycle Analysis Unsynchronized Cell Culture:

4x10⁵ HeLa Kyoto cells were seeded in in 6-well plates and incubated overnight. 30 min prior to the experiment, medium was changed to fresh growth medium with or without 10 µM ABT888. BPDE treatment (0.1, 0.5, 1 and 2 µM BPDE) was performed in incomplete medium at 37°C for 1 h.

Thereafter, medium was changed back to full growth medium and cells were cultured at 37°C for 24 hours. On the next day, cells were carefully washed with PBS, harvested with trypsin and pelleted by centrifugation (5 min; 1,000 rpm). The supernatant was discarded and the pellet resuspended in 300 µl cold PBS. The cell suspension was dropwise mixed with 700 µl ice-cold ethanol and kept for 20 min on ice or alternatively stored at -20°C. Cells were pelleted again by centrifugation (5 min; 200 xg; 4°C),

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washed with 150 µl cold PBS and again centrifuged (5 min; 300 xg; 4°C). This was repeated once, before the pellets were resuspended in 30 µl PBS and mixed thoroughly with 120 µl DNA extraction buffer (4 mM citric acid; 0.2 M Na2HPO4; pH 7.8). After 20 min under gentle agitation at 21°C on a thermal mixer, cells were centrifuged again (5 min; 300 xg) and resulting pellet was resolved in 200 µl DNA staining solution (20 µg/ml propidium iodide; 0.2 mg/ml DNase-free RNase A in PBS).

Incubation was performed for 30 min at RT before cells were stored on ice in the dark until measurement. Cell cycle phase was determined by analysis of cellular PI signal as a marker of DNA content. 30,000 cells were measured with a BD FACSCalibur and the CellQuest Pro 6.0 software. The obtained cytometric data were further analysed with FlowJo 8.8.7.

Synchronized Cell Culture:

Nocodazole interferes with the microtubules assembly, it binds to tubulin and blocks its polymerisation.

Thus it inhibits the formation of a functional spindle apparatus for mitosis. As a consequence the spindle checkpoint is activated and cells arrest in G2/M (prometaphase). During M phase, cells undergo dramatic morphological changes, they become more spherical and are thus only loosely attached to the growth surface. After 12 hours of nocodazole treatment most cells were arrested in G2/M and could be further synchronized by mitotic shake off.

For cell cycle synchronization 3.2x10⁶ HeLa Kyoto cells were seeded in T-75 cell culture flasks and incubated overnight. On the next day, 500 nM nocodazole was added to the normal growth medium to arrest cells in G2/M-phase. After 12 hours at 37°C most of the cells were stalled in this cell cycle phase.

To further increase the degree of synchronization a mitotic shake-off was performed, in which the easily detachable M-phase cells were separated from cells in different cell cycle phases. The M-phase cells containing supernatant was taken off, centrifuged (5 min; 200 xg) and washed twice in PBS. 4x10⁵ cells were seeded in 6-well plates (t0) and kept at 37°C. 10 min prior to BPDE treatment, the medium was changed to DMEM with or without 10 µM ABT888. 10 hours after seeding (t10) BPDE treatment was performed in incomplete medium at 37°C. Cells were allowed to recover in growth medium. At t11, t14, t18, t24 t26, t29 and t32 cells were washed with PBS, harvested and prepared for cell cycle analysis as described for unsynchronized cells.

3.2.5.14.3Cell Death Analysis (Annexin V/PI)

3x10⁵ HeLa Kyoto and HeLa Kyoto PARP-1 KO1 cells were seeded in 6-well plates and incubated overnight at 37°C. PARP inhibition, if desired, was performed with 10 µM ABT888 30 min before the experiment in incomplete medium. Cells were treated with BPDE in incomplete medium in the presence or absence of the PARP inhibitor (1, 2, 5 and 10 µM BPDE). Camptothecin (50-100 µM) was used as a positive control of cell death induction. After 1 hour the toxicants were removed and the cells were further cultured in growth medium (w/ or w/o ABT888) for 48 h. Then, medium of the samples was taken off and collected in a 50 ml centrifugation tube. Cells were washed with PBS and harvested with trypsin, both of which were subsequently pooled with the medium. The cell suspension was centrifuged with 1,000 rpm for 5 min and the resulting pellet resolved in cold PBS. Using the Casy Cell Counter, the total cell number was determined. 1x10⁶ cells were centrifuged (5 min; 1,000 xg; 4°C), the supernatant discarded and the cells resuspended in 1 ml annexin binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2). To 195 µl of cell suspension 5 µl annexin V-FITC was added and incubated in the dark for 15 min. Subsequently, 200 µl PI staining solution (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2; 10 µg/ml PI) were added and kept on ice until measurement.

Unstained, as well as PI and annexin V single-stained samples were prepared as well to establish correct gating and fluorescence compensation. Measurement of 20,000 cells was performed with a BD FACSCalibur and the CellQuest Pro 6.0 software and results were analysed with FlowJo 8.8.7.

- 79 - 3.2.5.15 alamarBlue Assay

The alamarBlue assay is a sensitive method based on the cellular reduction of resazurin to resorufin.

This causes a change in the fluorescence spectrum, resorufin is a detectable bright-red fluorescent dye.

The reduction of resazurin takes place in viable cells, using their endogenous redox system by oxidizing NADH to NAD⁺. Thus, the amount of reduced resazurin is an indicator of the cellular redox potential and as a consequence the cellular health. From the amount of fluorescence produced it can be inferred to the number of metabolizing and healthy cells.

After harvesting HeLa Kyoto or HeLa Kyoto PARP-1 KO1/KO2 cells with trypsin, cells were counted thrice and diluted to 6x10⁴ cells/ml. 100 µl/well (6,000 cells) of the cell suspension was distributed to a 96-well plate in technical trip- or quadruplicates and incubated for 3 hours at 37°C to give the cells the chance to adhere. 30 minutes prior to the cell treatment, medium was exchanged to fresh growth medium with or without 10 µM of the PARP inhibitor ABT888. As a positive control cells were incubated in 1 mM H2O2 in PBS/ 1 mM MgCl2 for 5 min at 37°C. For BPDE treatment, 0.01 – 10 µM BPDE were directly before usage diluted in prewarmed, incomplete DMEM. After 1 hour at 37°C medium was exchanged again to fresh growth medium. In case of PARP inhibition, ABT888 was present during and after the BPDE treatment. Cells were incubated for either 24 or 45 hours before 10 % alamarBlue solution was added to each well. After additional 4 hours, in which the healthy portion of the cells reduced resazurin to resorufin, the fluorescence was measured with an Varioskan Flash

After harvesting HeLa Kyoto or HeLa Kyoto PARP-1 KO1/KO2 cells with trypsin, cells were counted thrice and diluted to 6x10⁴ cells/ml. 100 µl/well (6,000 cells) of the cell suspension was distributed to a 96-well plate in technical trip- or quadruplicates and incubated for 3 hours at 37°C to give the cells the chance to adhere. 30 minutes prior to the cell treatment, medium was exchanged to fresh growth medium with or without 10 µM of the PARP inhibitor ABT888. As a positive control cells were incubated in 1 mM H2O2 in PBS/ 1 mM MgCl2 for 5 min at 37°C. For BPDE treatment, 0.01 – 10 µM BPDE were directly before usage diluted in prewarmed, incomplete DMEM. After 1 hour at 37°C medium was exchanged again to fresh growth medium. In case of PARP inhibition, ABT888 was present during and after the BPDE treatment. Cells were incubated for either 24 or 45 hours before 10 % alamarBlue solution was added to each well. After additional 4 hours, in which the healthy portion of the cells reduced resazurin to resorufin, the fluorescence was measured with an Varioskan Flash

Im Dokument The Role of Poly(ADP-Ribosyl)ation in the Molecular and Cellular Response to Nucleotide Excision Repair DNA-Lesions (Seite 86-0)