Supplementary Materials and Methods
Cell culture
The human osteosarcoma cell line U2OS were cultured in DMEM (Invitrogen)
supplemented with 10% FBS and the human non-small cell lung cancer cell line
H1299 in RPMI (Roswell Park Memorial Institute; Invitrogen) supplemented with
10% FBS. Both cell lines were obtained from the American Type Culture Collection
and maintained at 37°C under 5% CO2.
Transfection, cell treatments and flow cytometry
H1299 cells were transfected with a set of four small interfering RNA (siRNA)
duplexes directed against CK2, CK2’ and CK2 (ON-TARGET plus
SMARTpools, Dharmacon), respectively, or a ON-TARGET plus non-targeting pool
(Scrambled) using Lipofectamine2000 (Invitrogen) for 72 hours according to the
manufacturer's instructions. Where indicated, siRNA against DNA-PKcs (Santa Cruz
Biotechnology) were included (or Scrambled as control).
To analyze cell death, cells were incubated with 0.5 µM SYTOX green nucleic acid
stain for 10 minutes at 37°C, harvested by trypsinization, and combined with floating
cells present in the medium. Cells were resuspended in PBS containing 1% FBS. The
fraction of SYTOX green–positive cells was measured using a FACSCalibur (Becton
Dickinson) flow cytometer. 10,000 events were analyzed using the FL-1H filter for
determination of SYTOX green-positive cells. The acquired data were analyzed using
Cell Quest Pro software.
To test the effectiveness of zVAD(OMe)-fmk and zFA-fmk at the indicated
concentrations, U2OS cells were incubated with 50 µM Cisplatin (Sigma) for 24h.
Where indicated, 5 µM zVAD-fmk or 85 µM zFA-fmk were added. Cell death was
analyzed by SYTOX green staining as described above. Autophagy was analyzed
incubating cells with 0.5 mM monodansylcadaverine (MDC) for 1h at 37°C. For flow
cytometry analyses, cells were trypsinized and combined with floating cells from the
medium. The cells were resuspended in PBS containing 1% FBS and 10,000 cells
were analyzed using the FL-1 filter. For microscopic analyses, cells were grown on
coverslips and after incubation with MDC as described above the coverslips were
washed with PBS and analyzed on a DMRBE microscope (630x magnification)
equipped with a Leica DFC420C camera. As a positive control for autophagy M059K
cells were incubated 24h with 10 µM tamoxifen.
High salt protein extract and DNA-PK kinase assay
The activity of DNA-PK was measured in high-salt extracts. To make the extract,
cells were scraped and washed twice with ice-cold PBS prior to two washes in icecold Low Salt Buffer (10 mM HEPES pH 7.4, 25 mM KCl, 10 mM NaCl, 1.1 mM
MgCl2, 0.1 mM EDTA, 0.1 mM DTT). The cells were resuspended in 50 µl low salt
buffer (containing protease inhibitor cocktail (Complete, Roche) and 100 nM okadaic
acid), incubated on ice for 5 minutes and then frozen in a dry ice/ethanol bath for 5
minutes. The cell lysate was quick thawed in a 37°C water bath and mixed gently
with 5.5 µl high salt extraction buffer (5 M NaCl, 100 mM MgCl2, 10 mM DTT).
After 5 minutes incubation on ice, the extract was centrifuged and the pellet washed
with 25 µl salt wash buffer (10 mM HEPES pH 7.4, 25 mM KCl, 0.5 M NaCl, 10 mM
MgCl2, 0.1 mM EDTA, 1 mM DTT).
The activity of DNA-PK was measured in a total volume of 20 µl containing: 25 mM
HEPES pH 7.5, 20 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.01 mg/ml
sonicated salmon sperm, 250 µM synthetic peptide [EPPLSQEAFADLWKK or as a
control EPPLSEQAFADLWKK (both from EZ Biolab Inc, Carmel, IN, USA)], 125
µM cold ATP and 1 µCi [-32P]ATP (Hartmann Analytic, Braunschweig, Germany).
5 µg high-salt extract was added (in 2 µl) and incubated 5 minutes at 30°C. The tubes
were allowed to cool on ice and 10 µl of each reaction were spotted on P81
phosphocellulose paper (Whatmann). The paper squares were washed 3x5 minutes in
15% acetic acid and 1x1 minute in acetone before counting in a liquid scintillation
counter (Canberra Packard, Downers Grove, IL, USA).
Immunofluorescence
Cells grown on coverslips were rinsed with PBS and fixed for 20 min at room
temperature in 4% paraformaldehyde. After washing in PBS cells were permeabilized
by incubation for 2 min at 4°C with 0.1% Triton X-100 and 0.1% sodium citrate pH
7.0. Cells were blocked for 1h at room temperature in PBS supplemented with 0.2%
casein and 0.1% Tween20 (blocking buffer). They were, then, incubated overnight at
4°C with rabbit polyclonal anti-H2AX (p-S139) antibody (1:100; Cell Signaling
Technology). After extensive washing in blocking buffer, cells were incubated for 1h
with anti-rabbit IgG biotin-conjugated antibody (Dako) at a 1:300 dilution. Cells were
washed in PBS and incubated with FITC-conjugated streptavidin (Dako) at a dilution
1:50 for 30 min at room temperature. Finally, cells were washed in PBS and
counterstained with 4', 6'-diamidino-2-phenylindole (DAPI, Sigma) for 5 min at room
temperature. Cells were then washed in PBS, mounted onto slides with mounting
medium and subsequently analyzed on a Leica DMRBE fluorescence microscope
(630x magnification) equipped with a Leica DFC420C camera and processed using
ImageJ software (NIH) where 500 cells from each time point were analyzed.
In situ PLA
Glioblastoma cells grown on coverslips were fixed, permeabilized and blocked as
described above. Afterwards, cells were incubated overnight at 4°C with monoclonal
anti-DNA-PKcs antibody (1:50; Santa Cruz Biotechnology) and a rabbit polyclonal
antibody against CK2’ (1:10). For positive controls, cells were incubated with
monoclonal anti-DNA-PKcs antibody and rabbit polyclonal antibodies against either
KU70 or KU86 (both diluted 1:50, Santa Cruz Biotechnology). As a negative control,
M059J cells were used as they lack DNA-PKcs expression. The proximity ligation
assay [a fluorescent signal is generated only when two PLA probes are in close
proximity (<40 nm)] was performed using the DuoLinkTM Detection Kit 563. After
incubation with the primary antibodies, the coverslips were washed in blocking buffer
and incubated for 1h at 37°C with a pair of secondary antibodies conjugated with
oligonucleotides (PLA probes). Hybridization, ligation, amplification and detection
were performed according to the manufacturer’s instructions (Olink Biosciences).
Cell nuclei were visualized by Hoechst staining contained in the detection buffer and
the coverslips were mounted in DuoLink mounting medium and analyzed by a
fluorescent Leica DMRBE microscope equipped with a Leica DFC420C camera and
processed
using
the
freeware
software
Blobfinder
(http://www.cb.uu.se/∼amin/BlobFinder). An average analysis was made from all
signals deriving from one image divided by the number of cells in the image, for
obtaining the average signals/cell. For each sample 100 cells were analyzed.
In vivo DNA end-joining assay
The plasmid-based end-joining assay was performed as previously described (Wang
et al., 2006). As a reporter plasmid pEGFP-N1 (Clontech-Takara Bio Europe) was
used. It was digested overnight with HindIII (Fermentas), which cuts between the
promoter and the coding region of GFP. Complete digestion was verified by agarose
gel electrophoresis and the linear DNA fragment was purified using a gel extraction
kit (Qiagen). M059K cells transfected with siRNA against CK2 or CK2’ using
Dharmafect I were electroporated (NEONTM Tranfection system, Invitrogen), 48h
after siRNA transfection, with linear or circular supercoiled pEGFP-N1 according to
the manufacturer’s instructions (1 pulse, a pulse width of 20 and a pulse voltage of
1400 V). 24h after electroporation cells were trypsinized, and resuspended in PBS
containing 1% FBS and the percentage of GFP-positive cells determined (FL-1H).
For each sample 10,000 cells were counted and the acquired data were analyzed using
Cell Quest Pro software. The end-joining activity was calculated as the ratio between
GFP-positive cells following electroporation with linear pEGFP-N1 and the amount
of GFP-positive cells after electroporation with the circular supercoiled pEGFP-N1.
In vitro phosphorylation assay of KU70/86
1 pmol human recombinant CK2 was employed in an enzyme assay performed for
30 minutes at 30°C in a 20 µl reaction mixture containing 50 mM Tris pH 8.0, 10 mM
MgCl2, 1 mM DTT, 100 µM ATP, 1 µCi [-32P]ATP and 11 pmol GST-KU70, GSTKU86 (both from Abnova, Taipei, Taiwan) or, as a control, GST. Reactions were
terminated by addition of sample buffer and boiling for 5 min. Proteins were
separated by SDS-PAGE and revealed by staining the gel with Coomassie stain. The
incorporated radioactivity was detected by autoradiography.