www.sciencemag.org/cgi/content/full/340/6135/981/DC1
Supplementary Material for
Identification of a Heteromeric Complex That Promotes DNA Replication Origin Firing in Human Cells
Dominik Boos, Mona Yekezare, John F. X. Diffley*
*Corresponding author. E-mail: [email protected]
Published 24 May 2013, Science 340, 981 (2013)
DOI: 10.1126/science.1237448
This PDF file includes:
Materials and Methods
Figs. S1 to S12
Tables
References
Supplementary Material Materials and Methods Figures S1-S12 Tables S1
Materials and Methods
Tissue culture
Stable MTBP-Flag-AcGFP expressing HeLa-Kyoto cells were generated using
pIRESpuro3-Flag-AcGFP carrying the ORF of human MTBP that had been made
siRNA resistant using site directed mutagenesis with the oligonucleotides
TCTGGGGAGCAGATTGTACAGAgGgaAaaGcaACtGgcCaaTGTTCAAGTTTTA
GCTTTGGAAGAATGC and
GAGGGTCCTCGGGACTCAAtTacCCtCCtCgaCgcCaaAGAATTGCTGAAGTAC
TTTACCTCAGATG (small letters indicate silent mutations).
RNAi was carried out using RNAiMax (Invitrogen) according to the
manufacturer's instructions. In brief, 150000 HeLa-Kyoto, 200000 U2OS cells or
100000 HCT116 cells per 6 cm dish were transfected by reverse transfection
with 20 nM control siRNA (GL2/Dharmacon) or siRNA against MTBP (siNo1: 1:1
mix of oligonucleotides tcacattgttggatgctaa and gagagaaacagttagctaa (from
Dharmacon smart pool L-013953-00-0005); siNo2: Dharmacon ON-TARGET
siRNA UAAAUUGGAUGGAGCUAUU) or Treslin
(GAACAAAGGTTATCACAAA/Dharmacon). 10 µl (HeLa-Kyoto and HCT116) or
2
6 µl (U2OS) RNAiMax in 6 ml volume were used. A second forward transfection
was carried out 24h later for Treslin RNAis. Although RNAi of MTBP did not lead
to a reduction of Treslin levels in some experiments (Fig 3A) it had a minor effect
on Treslin/TICRR levels in a subset of experiments using Hela-Kyoto (fig. S12)
and U2OS cells (fig. S6). In contrast, Treslin/TICRR-RNAi severely co-depleted
MTBP in all cell types tested (fig. S12).
For cell synchronisations U2OS cells were blocked in 2 mM thymidine (Thy) for
24 h. For subsequent G2/M arrests cells were released from the Thy-mediated
block for 3h before adding 50 ng/ml nocodazole (Noc), followed by inbubation for
12-16 h. To synchronously release cells from Noc-mediated blocks mitotic cells
were harvested by mitotic shake-off, washed in PBS and re-plated. For
synchronous release from G1/S arrests cells were released from the first Thy
block for 14 h before adding 2 mM Thy a second time. Cells were grown for 22 h
before washing with PBS and release into fresh medium.
The Mdm2 inhibitor nutlin-3 (Sigma N6287) was used at the indicated
concentrations (typically 10 µM).
Live cell microscopy
For live cell microscopy AcGFP-Flag-PCNA carrying HeLa-Kyoto cells were
used as described (1). Cells were imaged with 20 min time frames. For
3
quantitative analyses the start and stop of S phase were defined as the time
point when S phase-like nuclear PCNA patterns became visible or disappeared,
respectively. PCNA patterns typical for early, mid and late S phase were readily
discernable in control cells by manual inspection of the movies. In the context of
on-going S phase active replication was also obvious in MTBP-RNAi treated
cells. Due to low replication efficiency in these cells this became more difficult to
judge towards the end of S phase when very few PCNA dots were left. Whenever
doubtful, for example when just a single dot lingered for extended periods of
time, the respective cell was not considered because in these cases we felt the
likelihood was high of mistaking PCNA-involving repair processes for genuine
replication, which cannot be distinguished by our analysis. G2 phases were
counted from the stop of S phase until nuclear envelope breakdown, judged by
re-distribution of GFP-PCNA from primarily nuclear throughout the whole cell. G1
phases were defined as cytokinesis until the start of S phase. RNAi-treated cells
were imaged from 24h after the start of transfection for 24h. Longer imaging
caused photodamage to cells. Because S phases of MTBP and Treslin siRNA-
treated cells were extremely long many S phases did not stop within this 24 h
time frame. Therefore, S phases had to be quantified as those longer or shorter
than 10 h rather than as duration of S in hours. In contrast, G1 and G2 phases
were short enough to be quantified as duration in hours.
4
Analysis of RNAi treated cells by Flow Cytometry and Chromatin
Fractionation
Staining of RNAi treated cells with BrdU (5-bromo-2'-deoxyuridine) and PI
(propidium iodide) followed by flow cytometry were described, as was the
analysis of protein levels after RNAi in whole cells (2).
For analysis of reduction of BrdU incorporation upon RNAi treatment S phase
cells were gated in BrdU-PI dot plots and displayed in BrdU-histograms. For
quantitative representation in column diagrams the relative BrdU fluorescence
was calculated by normalising the BrdU signal of S phase cells to the signal
intensity of BrdU-negative G1 and G2/M cells. This was normalised on BrdU
incorporation of control siRNA treated S phase cells calculated the same way.
Isolation of chromatin-enriched cell fractions was performed as described (3).
Cytosolic contamination was excluded by immunoblots for Tubulin whereas
chromatin enrichment was confirmed by specific isolation of histones, both
relative to whole cell extracts.
MTBP-Treslin-TopBP1 Binding Studies
Generation of cell lysates for immunoprecipitation (IP) and IP procedures of
endogenous MTBP, Treslin and TopBP1 were done as described (2), as were
5
binding studies using proteins overexpressed in 293T cells as well as in vitro
translated proteins (2). For detection of endogenous MTBP in
immunoprecipitates by immunoblotting a mouse anti-MTBP antibody was used to
avoid interference of rabbit IgG used for immunoprecipitations with detection by
HRP-coupled secondary anti-rabbit antibodies. However, only the rabbit anti-
MTBP-102-515 antibody was sensitive enough to reliably detect endogenous
MTBP in cell lystates. Therefore, for Figures 1A, 1B and 2E two different
antibodies had to be used to detect MTBP in lysates and Immuniprecipitates.
For pulldowns of in vitro translated Treslin fragments with MTBP C-terminally
Myc-tagged MTBP (Origene, RC204388) was expressed in 293T cells and anti-
Myc antibody containing beads were loaded by incubation in the respective
lysate containing MTBP or not (for control precipitations). All MTBP-Treslin
binding experiments were done in 20 mM Hepes 8.0, 200 mM NaCl, 0.1 % Triton
X-100, 5 mM MgCl2, 5 mM 2-mercaptoethanol, 5 % Glycerol, Complete EDTA-
free protease Inhibitor Cocktail (Roche), 5 µg/ml Cytochalasin D.
Treslin/TICRR mutants
Treslin/TICRR mutants carried the following alterations: ΔCIT, deletion of
amino acids 1-264; ΔM1, deletion of amino acids 265-408; ΔM2, deletion of
amino acids 409-593; ΔSTD, deletion of amino acids 594-880; 2PM, mutation of
6
threonine 969 and serine 1001 to alanine (2); ΔCT, deletion of C-terminal 854
amino acids.
Fiber Analysis
Cells were labeled with 25uM IdU (5-Iodo-2′-deoxyuridine), followed by labeling
with 250uM CldU (5-Chloro-2′-deoxyuridine). DNA fiber spreading and staining
were performed as described by Merrick et al. (4).
Analysis of AcGFP-Treslin Localization in Cells by Immunofluorescence
Control U2OS cells or cell lines stably expressing Treslin-WT or the indicated
mutants were fixed with 4% paraformaldehyde in PBS for 10 min, incubated in
PBS/0.1% Triton X-100, blocked in 1% BSA in TBS/0.01% Tween for 30 min,
and then incubated in chicken anti-GFP antibody (1/1000 dilution) in TBS/0.01%
Tween /1% BSA for 1h. After three washes in PBS/0,01% Triton cells were
incubated in Alexa-Fluor 488 goat anti-chicken antibody (1/1000 dilution) and 0.2
µg/ml DAPI (SIGMA) before washing another three times, and then analysed by
fluorescence microscopy. Images of different cell lines were treated exactly the
same way using the Photoshop software (Adobe) to ensure comparability.
7
Antibodies and other materials
Anti-MTBP: affinity purified rabbit polyclonal antibodies against hMTBP amino
acids 102-515 (immunoblots) and anti-hMTBP amino acids 1-284
(immunoprecipitations); mouse anti-MTBP clone PHL-1 (abcam); anti-Treslin:
rabbit polyclonal anti-hTreslin-1566-1910, affinity purified; anti-GFP: mouse
monoclonal, clone JL-8 (Clontech/632381); anti-TopBP1: rabbit polyclonal
antibodies: rabbit polyclonal anti-TopBP1-1-360 (immunoblots) and anti-TopBP1-
430-800 (immunoprecipitations), affinity purified; anti-BrdU-FITC: mouse
monoclonal (BD Pharmingen/556028); anti-Myc: mouse monoclonal 9E10; anti-
Mcm2: goat polyclonal (Santa Cruz; sc-6680); anti-Tubulin: mouse anti-alpha-
Tubulin (Sigma; T5168); anti-p53: mouse anti-p53 (Calbiochem; OP43); anti-p21:
mouse anti-p21 (clone AC8.3); anti-PCNA: mouse anti-PCNA clone PC10; anti-
Mdm2: mouse anti-Mdm2 clone SMP-14; anti-Psf3: mouse monoclonal
(generous gift of Juan Méndez); anti-Cdc45: rat anti-Cdc45 clone 3G10
(generous gift from HP Nasheuer and F. Grosse and Helmut Pospiech); anti-
Cyclin A: rabbit polyclonal (Santa Cruz; H432); anti-BrdU: mouse anti-BrdU (IdU
detection in DNA fibre labelling; BD Biosciences, Clone 44, 347580); rat anti-
BrdU (CldU detection in DNA fibre labelling; Abcam, ab3626); chicken anti-GFP
antibody (Abcam 13970); Alexa-Fluor 488 goat anti-chicken antibody (Molecular
Probes; A11039)
Protein G Sepharose 4 Fast Flow (GE Healthcare/17-0618-01),
8
Legends for Figures S1-12 and Table S1
Fig. S1:
Immunoprecipitation of GFP-Flag-Treslin for mass spectrometric identification
of Treslin interactors. Native cell lysates of nocodazole-arrested control HeLa-
Kyoto cells or cells stably expressing AcGFP-Flag-Treslin (2) were used for
immunoprecipitation with anti-Flag sepharose (Sigma) followed by elution with
3xFag-peptide (Sigma). Whole lanes of a Coomassie stained SDS PAGE gel
were cut into strips and used for mass spectrometry. The results are shown in
Table S1.
Fig. S2
Large fractions of MTBP and Treslin form a complex in cell lysates. Native
HeLa cell extracts were immunodepleted of endogenous MTBP (i) or Treslin (ii).
Levels of Treslin, MTBP and Tubulin in the IP-flow-throughs were compared by
immunoblotting to those in flow-throughs of decreasing amounts of control (ctr)-
IPs (100 – 12.5 %). Greater than 90% depletion of MTBP co-depletes around
50% of Treslin, and greater than 90% depletion of Treslin co-depletes 50%-75%
of MTBP.
Fig. S3
9
A Treslin protein fragment containing amino acids 260-671 is sufficient for
interaction with MTBP.
A) Schematic relating the Treslin/TICRR fragments used in fig. S3B to the
Treslin/TICRR domain structure. Start and stop of the fragments refer to
the position of the amino acids (aa) in the full-length Treslin/TICRR
protein. The Treslin-260-671 fragment contains the whole region deleted
in mutants ΔM1 and ΔM2. It also contains roughly 80 aa more at the C-
terminus to avoid deletion of any regions essential for the M-domain.
However, Treslin-260-671 clearly excludes the STD domain (5).
B) 35S-labelled in vitro translated Treslin/TICRR fragments (fr.) (fig. S3A)
were used for binding reactions (Pulldowns) with beads containing MTBP-
Myc (+) or control beads (-) as described in Materials and Methods. MTBP
was detected by anti-Myc immunoblotting, and Treslin/TICRR fragments
by autoradiography in pulldowns and reticulocyte lysates (Input).
Fig. S4
Treslin-WT, 2PM and ΔM2 localise to the nucleus in the cell lines used for Fig
2C and D. Fluorescence microscopy images of the indicated U2OS cell lines and
control U2OS cells analysed by anti-GFP immunofluorescence as described in
Materials and Methods are shown. DAPI staining (blue) indicates the DNA-
containing nucleus, anti-GFP staining visualises AcGFP-Flag-Treslin and merged
10
indicated nuclear GFP-signals. Anti-GFP antibodies had to be used because
AcGFP signals were too weak for reliable detection.
Fig. S5
Treatment of Hela cells with two independent siRNAs against MTBP inhibits
replication. Analysis of cells of Fig. 3A and B by pseudo coloured dot plots of PI
and BrdU fluorescence. 2.4x, 2.9x and 4.7x indicate times reduction of BrdU
incorporation of S phase cells compared to siCtr treated cells.
Fig. S6:
U2OS cells show the same reduction of replication phenotype upon depletion of
MTBP as HeLa-Kyoto cells demonstrating this phenotype is not cell type specific.
U2OS cells were treated with control siRNA (siCtr) or MTBP siRNA (siNo1 and
siNo2) and analysed for replication efficiency and cell cycle distribution as
described for HeLa-Kyoto cells in figures 3A and B.
Fig. S7:
Replication in MTBP-RNAi treated cells can be fully rescued by expression of
siRNA-resistant MTBP-Flag-GFP. The rescue is dependent on expression of
stable MTBP but independent of the cell clone used.
11
A) Endogenous but not transgenic MTBP is eliminated by RNAi against
MTBP. A HeLa-Kyoto cell clone independent from the one used for Fig. 3
C and D expressing siRNA-resistant MTBP-Flag-GFP or control cells were
treated with siNo2 against MTBP and analysed for levels of endogenous
and transgenic MTBP as described in Fig. 3C.
B) Rescue of MTBP-RNAi-mediated suppression of replication using an
MTBP-Flag-AcGFP expressing cell clone independent of the one used for
Fig. 3 C and D. The cells of experiment A were analysed for replication
efficiency as in Fig 3D.
C) Virtually full rescue of replication upon knock-down of MTBP by stable
expression of siRNA-resistant MTBP-Flag-AcGFP using two independent
siRNAs. HeLa control cells and cells stably expressing siRNA-resistant
MTBP-Flag-AcGFP were treated with siRNA against MTBP (siNo1 or
siNo2) or control siRNA (siCtr) for 48 h. Analysis for replication efficiency
by BrdU incorporation and flow cytometry was done as described in
Materials and Methods. Quantification of at least three independent
experiments per condition are shown in a column diagram. Error bars
represent standard error of the mean.
12
Fig. S8:
In contrast to MTBP-Flag-AcGFP, N-terminally AcGFP-Flag tagged MTBP
does not interact with Treslin/TICRR and does not rescue replication upon
depletion of endogenous MTBP
A) MTBP-Flag-AcGFP, but not AcGFP-Flag-MTBP binds endogenous
Treslin/TICRR. Control cells or cells stably expressing either N- or C-
terminally tagged MTBP were used for IP with anti-MTBP or anti-GFP
antibodies. MTBP-IP immunoprecipitated all MTBP versions, whereas
GFP-IP only precipitated MTBP containing GFP on either end. Treslin co-
precipitated with all anti-MTBP-IPs and with the anti-GFP-IP from the cell
line expressing MTBP-Flag-AcGFP, but not from the AcGFP-Flag-MTBP
expressing line. This indicates the N-terminal tag interferes with Treslin
binding to MTBP.
B) Stable expression of MTBP-Flag-AcGFP, but not AcGFP-Flag-MTBP
rescues replication in cells depleted of endogenous MTBP. Control cells or
cells stably expressing either N- or C-terminally tagged MTBP were
depleted of endogenous MTBP using RNAi and analysed for replication
efficiency as in Fig. 3A and B. Control and AcGFP-Flag-MTBP expressing
lines showed almost the same degree of reduction of BrdU incorporation,
whereas MTBP-Flag-AcGFP expressing cells incorporated BrdU nearly as
13
efficiently as siCtr-treated cells. This indicates that AcGFP-Flag-MTBP is
not active in promoting replication.
Fig. S9:
Cells lacking Treslin have longer S phases and shorter G2 phases than control
cells. HeLa-Kyoto cells expressing GFP-PCNA were treated with Treslin or
control siRNA. Timing of cell cycle phases were analysed by time-lapse
microscopy as described for Fig. 3E and Materials and Methods. Long S phases
and short G2 phases are reminiscent of the phenotypes seen with cells depleted
of MTBP.
Fig. S10:
An active Mdm2-p53 pathway is dispensable for the replication defect upon
RNAi-mediated depletion of MTBP.
A) HCT116 cells show reduced replication upon MTBP knock down,
regardless of their p53 status. HCT116 p53+ (i) cells or HCT116-p53-/-
cells (ii) were depleted of MTBP using siNo1 or control siRNA (siCtr) for
62 h. Cells were analysed by immunoblotting for MTBP and Tubulin levels,
and for replication efficiency by incorporation of BrdU and flow cytometry
as described in Materials and Methods. S phase cells of both cell lines
show a reduction in BrdU incorporation.
14
B) The Mdm2 ubiquitin ligase-inhibitor nutlin-3 does not suppress the
replication defect upon MTBP depletion. HeLa-Kyoto cells (do not have an
active p53 pathway) and HCT116 p53-/- cells were depleted of MTBP in
the absence or presence of 10 µM of the Mdm2 ubiquitin ligase inhibitor
nutlin-3. Cells were then analysed by flow cytometry as described in
Materials and Methods for incorporation of BrdU. In both cell lines S phase
cells show a reduction of BrdU incorporation (1.9-fold for HeLa cells and
1.4 or 1.5-fold for HCT116 p53-/- cells) regardless of whether nutlin-3 was
present. Whereas it is formally possible that nutlin-3 is not active in HeLa-
Kyoto cells we can demonstrate that it is clearly active in HCT116 cells
(fig. S10C). Thus, MTBP’s activity in promoting replication is independent
of the ubiquititin ligase activity of Mdm2.
C) Nutlin-3 is active in HCT116 cells and induces a p53-mediated cell cycle
arrest. HCT116 p53-positive or p53-/- cells were treated with the indicated
concentrations of nutlin-3 for 48 h. Cells were analysed for cell cycle
distribution by chromatin staining with propidium iodide (PI) and flow
cytometry. p53-positive, but not p53-/- cells, show an efficient cell cycle
arrest in G1 and G2/M even with 3.3 µM concentration of nutlin-3. This
indicated that nutlin-3 inhibits Mdm2 in HCT116 cells in a p53-dependent
manner. Cell cycle quantifications represent Watson (Pragmatic) analyses
using the FlowJo Software.
15
Fig. S11:
Knocking down MTBP leads to a late p53 response in U2OS cells. U2OS cells
were treated with control siRNA (siCtr) or siRNA against MTBP. Levels of MTBP,
p53, p21 and Tubulin (Tub.) were analysed by immunoblotting. An induction of
p53 was observed specifically in the absence of MTBP. The induction of the p53
pathway was productive because the target p21 was induced too (62 h). p53
activation is apparent only in cells treated for 62 h but not in cells treated for 48 h.
By 48 h a strong replication phenotype has already been established (fig. S6),
indicating that p53 activation is likely a secondary response to the primary
inhibition of replication by MTBP depletion.
Fig. S12:
Treslin/TICRR is required to maintain MTBP protein levels in cells.
A) Treslin/TICRR was depleted (siTres) from HeLa-Kyoto control cells or
cells stably expressing siRNA-resistant AcGFP-Flag-Treslin using RNAi as
described (2). Whole cell lysates were compared with control RNAis
(siCtr) for levels of Treslin/TICRR, MTBP and Tubulin by immunoblotting.
Upon RNAi-depletion of Treslin/TICRR MTBP levels decreased
dramatically. Overexpression of siRNA-resistant AcGFP-Flag-Treslin was
sufficient to rescue levels of MTBP in Treslin-RNAi-treated cells.
16
B) Treslin/TICRR (siTres) or MTBP (siMTBP, siNo2) were depleted from
HeLa-Kyoto control cells or cells stably expressing siRNA-resistant MTBP-
Flag-AcGFP using RNAi and analysed as in A. Upon RNAi-depletion of
MTBP or Treslin/TICRR the level of the other protein decreased (lanes 1-
3). However, MTBP-depletion affected Treslin/TICRR levels much less
than Treslin/TICRR-depletion affected MTBP (see also long exposures).
Expression of RNAi-resistant MTBP-Flag-AcGFP rescued levels of Treslin
(lanes 3 and 6). Even the high amounts of transgenic MTBP-Flag-AcGFP
(compared to endogenous levels) decreased when Treslin was depleted
(lanes 4 and 5).
Table S1:
Mass spectrometry results of fig S1. Shown are the identified proteins, their
accession numbers, their molecular weights as well as the number of peptides
found in Flag-peptide eluates of control cells (Ctr) and GFP-Flag-Treslin
expressing cells (2) (see fig.S1).
1. S. Lekomtsev, J. Guizetti, A. Pozniakovsky, D. W. Gerlich, M. Petronczki, Evidence that the tumor-suppressor protein BRCA2 does not regulate cytokinesis in human cells. J Cell Sci 123, 1395 (May 1, 2010).
2. D. Boos et al., Regulation of DNA Replication through Sld3-Dpb11 Interaction Is Conserved from Yeast to Humans. Curr Biol 21, 1152 (Jul 12, 2011).
17
3. N. Mailand, J. F. X. Diffley, CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C–dependent proteolysis. Cell 122, 915 (2005).
4. C. J. Merrick, D. Jackson, J. F. X. Diffley, Visualization of altered replication dynamics after DNA damage in human cells. J Biol Chem 279, 20067 (May 7, 2004).
5. L. Sanchez-Pulido, J. F. X. Diffley, C. P. Ponting, Homology explains the functional similarities of Treslin/Ticrr and Sld3. Curr Biol 20, R509 (Jun 22, 2010).
Cells HeLa HeLa-GFP-Flag-Treslin
FLAG-IP/FLAG-peptide eluate
220
50
30
20
70
90120
GFP-Flag-Treslin
Coomassie staining
For mass spectrometry results see Suppl. Table 1
MW[kD]
Boos et al. Fig. S1
100
100
75 50 25 12.5Fraction
loaded [%]
CtrIPM
TBP
IP
Flow
Thr
ough
100
100
75 50 25 12.5
CtrIP
Treslin
MTBP
Loading(Tubulin)
Tres
lin IP
Fl
ow T
hrou
gh
(i) (ii)
Boos et al Fig. S2
MTBP+-+-+-+-1-437 1-671 1-837 260-671
1-43
7
PulldownsInputs
Tres. fr. (aa)
Treslin(Autorad.)
MTBP
B
1-67
1
1-83
726
0-67
1
Boos et al. Figure S3
PPCIT STD
PPSTD
Cdc45Dpb11Sld7
Sld3
Treslin
Treslinfragment
aa 1-437aa 1-671aa 1-837
aa 260-671
Sld3 interactors
1910 aa
668 aa
C-term.
A
ΔCITDel./Mut. ΔM1 ΔM2 ΔSTD 2PM ΔCT(see Fig. 2)
M dom.
DAPI anti-GFP merge
U2OS
WT
2PM
ΔM2
Boos et al. Fig. S4
PI [AU]
siCtr siMTBP
48h 62h48h
2.4x 2.9x
2.9x 4.7x
siNo2 siNo2
siNo1 siNo1Br
dU [A
U lo
g]
1C 2C 1C 2C 1C 2C
Boos et al Fig. S5
siCtr siMTBP
48h 62h48h
1.9x 2.2x1.9x
2.4x 3.6x2.4x
siCtr 48hsiMTBP 48hsiMTBP 62h
siNo2 siNo2
siNo1 siNo1
siNo2
siNo1
MTBP
Treslin
Loading(Ponc.)
siCtr
siNo1
48h
siNo2
62h
siCtr
siNo1
siNo2PI [AU] BrdU [AU log]1C 2C
BrdU
[AU
log]
rel.
cell
num
ber
phospho-Tres.
PI [AU]
S phase cells only
S phase cells only
U2OS cells
Boos et al. Fig. S6
HeLa-MTBP-GFP
siCtr
siNo2
HeLa
siCtr
siNo2
siNo2siCtr siNo2
HeLa-MTBP-GFP
HeLa
MTBP
Tubulin
MTBP-Flag-AcGFP
endogenous MTBP
propidium iodide
BrdU
[log
]
A
B
C
Boos et al. Fig. S7
siCtr siNo2 siNo2
HeLa-MTBP-GFP
HeLa
1.0
0
0.2
0.4
0.8
0.6
rel.
BrdU
inco
rp.
of S
pha
se c
ells
[AU
]
Kyoto siC
tr
Kyoto siM
TBP 2/4
Kyoto-MTB
P-GFP
siUTR
2789
Kyoto-MTB
P-GFP
siMTB
P2/4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
siNo1 siNo1
HeL
a
GFP
-MTB
P
MTB
P-G
FP HeLaGFP-MTBP
MTBP-GFP
MTB
PG
FP
MTB
PG
FP
MTB
PG
FP
IPsInputs
IP-antibody
Treslin
MTBPtransg.
endog.
2.5x 2.1x 1.1xHeLa GFP-MTBP MTBP-GFP
rel.
cell
num
ber
BrdU [AU log]
A
B siCtr siMTBP
S-phase cells only
S-phase cells only
S-phase cells only
Boos et al Fig. S8
Cell line
Cell line
Treslin
MTBP
GL241
440
2
4
6
2
4
0
6
GL2 siTres
G2 phase
GL2 siTresGL241
440
20
40
60
80
100
% c
ells
with
S lo
nger
than
10h
% S
pha
ses
long
er
than
10
h
S phase
A B
Dur
atio
n [h
]
Boos et al. Fig. S9
1.9x
1.9x
1.4x
1.5x
HeLa HCT116p53-/-
no Nut
+ Nut rela
tive
cell
coun
t
BrdU [log]
S phase cells only
S phase cells only
siCtr siNo1
B
HCT116p53+
siCt
r
siN
o1
MTBP
Loading(Tubulin)
siNo1 siCtr
A
S phase cells only
siNo1 siCtr
BrdU [log]
BrdU [log]
HCT116 p53-/-
siCt
r
siN
o1
MTBP
Loading(Tubulin)
S phase cells only
0 µM 3.3 µM 5 µM
HCT116 p53+ HCT116 p53 -/-
36%S
10% 4% 47% 46% 45%
45%G1
16%G2/M
67%
20%
74%
27%
10 µM 0 µM 3.3 µM 5 µM 10 µM
2%
70%
29%
27%
22%
25%
25%
28%
23%
G1 G2/M G1 G2/M G1 G2/M
46%
29%
23%
G1 G2/M
Nutlin
PIG1 G2/M G1 G2/M G1 G2/M G1 G2/M
PI
rela
tive
cell
coun
t
C
(i)
(ii)
Boos et al. Fig. S10
MTBP
p53
p21
Loading(Tub.)
cross-reac.cross-
reac.
62h RNAi
siCtr
siNo1
MTBP
p53
Loading(Tub.)
48h RNAi
siCtr
siNo1
Boos et al. Fig. S11
A
siCtr
siTre
ssiC
trsiT
res
HeLaHeLa-
GFP-Treslin
Treslin
MTBPLoading
(Tub.)
B
Treslin
MTBP
Loading(Tubulin)
HeLa HeLa-MTBP-GFP
siCt
r
siTr
es
siM
TBP
siCt
r
siTr
es
siM
TBP
transg.
endog.
1 2 3 4 5 6
Boos et al. Fig. S12
endog. long expos.
long expos.
Supplementary Table 1
# Identified Proteins (94) Accession Number Molecular Weight Ctr cells (peptide number) AcGFP-‐Flag-‐Treslin cells (peptide number)1 Myosin-‐9 OS=Homo sapiens GN=MYH9 PE=1 SV=4 MYH9_HUMAN (+1) 227 kDa 79 812 Treslin OS=Homo sapiens GN=TICRR PE=1 SV=2 TICRR_HUMAN 211 kDa 0 583 Kinesin-‐like protein KIF11 OS=Homo sapiens GN=KIF11 PE=1 SV=2 KIF11_HUMAN 119 kDa 35 284 Actin 5 OS=Aedes aegypti GN=act-‐5 PE=2 SV=1 Q4PKE5_AEDAE 42 kDa 9 105 Protein arginine N-‐methyltransferase 5 OS=Homo sapiens GN=PRMT5 PE=1 SV=4 ANM5_HUMAN 73 kDa 20 196 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6 K2C1_HUMAN (+1) 66 kDa 12 67 Epithelial protein lost in neoplasm beta variant (Fragment) OS=Homo sapiens PE=1 SV=1 Q59FE8_HUMAN 86 kDa 12 218 Heat shock 70kDa protein 1A OS=Homo sapiens GN=HSPA1A PE=2 SV=1 A8K5I0_HUMAN (+4) 70 kDa 8 139 Alpha-‐S2-‐casein OS=Bos taurus GN=CSN1S2 PE=1 SV=2 CASA2_BOVIN 26 kDa 0 010 Green fluorescent protein OS=Aequorea coerulescens PE=1 SV=1 Q6YGZ0_9CNID 27 kDa 0 611 Myosin-‐Ic OS=Homo sapiens GN=MYO1C PE=1 SV=4 MYO1C_HUMAN (+1) 122 kDa 15 2112 Spectrin beta non-‐erythrocytic 1 OS=Homo sapiens GN=SPTBN1 PE=2 SV=1 B2ZZ89_HUMAN (+1) 275 kDa 17 2313 Serum albumin OS=Bos taurus GN=ALB PE=1 SV=4 ALBU_BOVIN 69 kDa 7 614 cDNA FLJ53272, highly similar to Homo sapiens LIM domain 7 (LMO7), mRNA OS=Homo sapiens PE=2 SV=1 B7Z8W3_HUMAN 145 kDa 5 1815 Thyroid hormone receptor-‐associated protein 3 OS=Homo sapiens GN=THRAP3 PE=1 SV=2 TR150_HUMAN 109 kDa 14 616 Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1 HSP7C_HUMAN (+7) 71 kDa 7 1217 Vimentin variant (Fragment) OS=Homo sapiens PE=2 SV=1 Q53HU8_HUMAN (+2) 54 kDa 4 1218 Alpha S1 casein OS=Bos taurus GN=CSN1S1 PE=2 SV=1 B5B3R8_BOVIN (+1) 24 kDa 0 019 Major allergen beta-‐lactoglobulin OS=Bos taurus PE=2 SV=1 B5B0D4_BOVIN 20 kDa 0 020 cDNA, FLJ96568, highly similar to Homo sapiens tropomyosin 3 (TPM3), mRNA OS=Homo sapiens PE=2 SV=1 B2RDE1_HUMAN (+4) 29 kDa 8 921 Keratin, type I cytoskeletal 18 OS=Homo sapiens GN=KRT18 PE=1 SV=2 K1C18_HUMAN (+1) 48 kDa 13 822 Mdm2-‐binding protein OS=Homo sapiens GN=MTBP PE=1 SV=1 MTBP_HUMAN 102 kDa 0 1323 Uncharacterized protein OS=Bos taurus PE=4 SV=1 E1BIM4_BOVIN 18 kDa 0 024 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6 K1C10_HUMAN 59 kDa 5 125 Kappa casein (Fragment) OS=Bos taurus PE=4 SV=1 A3FJ56_BOVIN (+16) 18 kDa 0 026 Coronin-‐1C_i2 protein OS=Homo sapiens GN=CORO1C PE=2 SV=1 A7MAP0_HUMAN (+5) 54 kDa 4 527 Shootin-‐1 OS=Homo sapiens GN=KIAA1598 PE=1 SV=4 SHOT1_HUMAN 72 kDa 7 828 Methylosome protein 50 OS=Homo sapiens GN=WDR77 PE=1 SV=1 MEP50_HUMAN (+3) 37 kDa 3 229 60S ribosomal protein L7 OS=Homo sapiens GN=RPL7 PE=1 SV=1 RL7_HUMAN 29 kDa 4 630 Vasodilator-‐stimulated phosphoprotein OS=Homo sapiens GN=VASP PE=1 SV=3 VASP_HUMAN 40 kDa 6 431 Uncharacterized protein OS=Homo sapiens GN=RPL4 PE=4 SV=1 E7EWF1_HUMAN (+3) 46 kDa 8 532 Nucleolin, isoform CRA_c OS=Homo sapiens GN=NCL PE=2 SV=1 B3KM80_HUMAN (+6) 59 kDa 8 933 Heterogeneous nuclear ribonucleoprotein U OS=Homo sapiens GN=HNRNPU PE=1 SV=6 HNRPU_HUMAN (+3) 91 kDa 4 234 Ribosomal protein S6 OS=Homo sapiens GN=RPS6 PE=2 SV=1 A2A3R6_HUMAN (+11) 29 kDa 4 635 EF-‐hand domain-‐containing protein D2 OS=Homo sapiens GN=EFHD2 PE=1 SV=1 EFHD2_HUMAN 27 kDa 5 536 Protein enabled homolog OS=Homo sapiens GN=ENAH PE=1 SV=2 ENAH_HUMAN (+2) 67 kDa 0 637 Drebrin OS=Homo sapiens GN=DBN1 PE=1 SV=4 DREB_HUMAN (+2) 71 kDa 1 1038 Tubulin alpha-‐1C chain OS=Homo sapiens GN=TUBA1C PE=1 SV=1 TBA1C_HUMAN (+22) 50 kDa 4 739 Spectrin alpha chain, brain OS=Homo sapiens GN=SPTAN1 PE=1 SV=3 SPTA2_HUMAN (+1) 285 kDa 6 740 Heat shock protein HSP 90-‐beta OS=Homo sapiens GN=HSP90AB1 PE=1 SV=4 HS90B_HUMAN (+8) 83 kDa 11 241 Serine/threonine-‐protein kinase 38-‐like OS=Homo sapiens GN=STK38L PE=1 SV=3 ST38L_HUMAN (+5) 54 kDa 5 542 Uncharacterized protein OS=Canis familiaris GN=TPM4 PE=3 SV=1 E2R661_CANFA 29 kDa 4 443 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7 K2C8_HUMAN (+2) 54 kDa 5 344 Tubulin beta chain OS=Homo sapiens GN=TUBB PE=1 SV=2 TBB5_HUMAN (+20) 50 kDa 5 845 40S ribosomal protein S3a OS=Homo sapiens GN=RPS3A PE=1 SV=2 RS3A_HUMAN (+8) 30 kDa 2 846 60S ribosomal protein L7a OS=Homo sapiens GN=RPL7A PE=1 SV=2 RL7A_HUMAN (+16) 30 kDa 5 347 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3 K1C9_HUMAN 62 kDa 2 148 Elongation factor 1-‐alpha OS=Reclinomonas americana PE=2 SV=1 C1K9U5_RECAM (+4) 49 kDa 4 349 Protein flightless-‐1 homolog OS=Homo sapiens GN=FLII PE=1 SV=2 FLII_HUMAN (+4) 145 kDa 3 350 Protein phosphatase 1B OS=Homo sapiens GN=PPM1B PE=1 SV=1 PPM1B_HUMAN (+9) 53 kDa 1 051 40S ribosomal protein S3 OS=Homo sapiens GN=RPS3 PE=1 SV=2 RS3_HUMAN (+5) 27 kDa 2 252 Nucleolar RNA helicase 2 OS=Homo sapiens GN=DDX21 PE=1 SV=5 DDX21_HUMAN 87 kDa 2 553 14-‐3-‐3 protein epsilon OS=Homo sapiens GN=YWHAE PE=1 SV=1 1433E_HUMAN (+16) 29 kDa 0 554 Uncharacterized protein OS=Homo sapiens GN=MYO6 PE=4 SV=1 E7EW20_HUMAN (+1) 149 kDa 1 555 Stress-‐70 protein, mitochondrial OS=Homo sapiens GN=HSPA9 PE=1 SV=2 GRP75_HUMAN (+10) 74 kDa 4 556 Polyadenylate-‐binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2 PABP1_HUMAN (+13) 71 kDa 5 457 Ribosomal protein S2 OS=Homo sapiens GN=RPS2 PE=2 SV=1 Q3KQT6_HUMAN (+1) 31 kDa 3 558 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2 K22E_HUMAN (+1) 65 kDa 8 059 Plectin 1, intermediate filament binding protein 500kDa, isoform CRA_b OS=Homo sapiens GN=PLEC1 PE=4 SV=1D3DWL0_HUMAN (+1) 234 kDa 1 860 Uncharacterized protein C19orf21 OS=Homo sapiens GN=C19orf21 PE=1 SV=1 CS021_HUMAN 75 kDa 1 561 60S ribosomal protein L8 OS=Homo sapiens GN=RPL8 PE=1 SV=2 RL8_HUMAN (+13) 28 kDa 1 162 BTB/POZ domain-‐containing protein KCTD2 OS=Homo sapiens GN=KCTD2 PE=1 SV=3 KCTD2_HUMAN (+6) 29 kDa 2 163 Spindlin, isoform CRA_a OS=Homo sapiens GN=SPIN PE=2 SV=1 A8K0X6_HUMAN (+5) 30 kDa 2 064 Splicing factor 3B subunit 1 OS=Homo sapiens GN=SF3B1 PE=1 SV=3 SF3B1_HUMAN (+4) 146 kDa 3 065 E3 ubiquitin-‐protein ligase TRIM21 OS=Homo sapiens GN=TRIM21 PE=1 SV=1 RO52_HUMAN 54 kDa 0 266 Isoleucyl-‐tRNA synthetase, cytoplasmic variant (Fragment) OS=Homo sapiens PE=2 SV=1 Q59G75_HUMAN (+2) 146 kDa 5 067 RPS4X protein (Fragment) OS=Homo sapiens GN=RPS4X PE=2 SV=2 Q96IR1_HUMAN (+24) 27 kDa 1 068 78 kDa glucose-‐regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2 GRP78_HUMAN (+27) 72 kDa 2 269 Glutaminyl-‐tRNA synthetase OS=Homo sapiens GN=QARS PE=1 SV=1 SYQ_HUMAN (+4) 88 kDa 4 070 Cell division cycle 5-‐like protein OS=Homo sapiens GN=CDC5L PE=1 SV=2 CDC5L_HUMAN (+10) 92 kDa 2 171 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 PE=1 SV=5 K2C7_HUMAN (+1) 51 kDa 0 372 Heat shock protein HSP 90-‐alpha OS=Homo sapiens GN=HSP90AA1 PE=1 SV=5 HS90A_HUMAN (+9) 85 kDa 3 073 Alpha-‐enolase OS=Homo sapiens GN=ENO1 PE=1 SV=2 ENOA_HUMAN (+2) 47 kDa 3 074 Fatty acid synthase OS=Homo sapiens GN=FASN PE=1 SV=3 FAS_HUMAN 273 kDa 3 075 OTU domain-‐containing protein 4 OS=Homo sapiens GN=OTUD4 PE=1 SV=3 OTUD4_HUMAN 124 kDa 3 076 Gelsolin (Amyloidosis, Finnish type) OS=Homo sapiens GN=GSN PE=2 SV=1 A2A418_HUMAN (+12) 81 kDa 2 077 Bcl-‐2-‐associated transcription factor 1 OS=Homo sapiens GN=BCLAF1 PE=1 SV=2 BCLF1_HUMAN 106 kDa 2 078 CYTSA protein OS=Homo sapiens GN=CYTSA PE=2 SV=1 B2RMV2_HUMAN (+12) 125 kDa 0 179 CCR4-‐NOT transcription complex subunit 1 OS=Homo sapiens GN=CNOT1 PE=1 SV=2 CNOT1_HUMAN (+6) 267 kDa 0 180 Cyclin-‐dependent kinase 2 OS=Homo sapiens GN=CDK2 PE=1 SV=2 CDK2_HUMAN (+26) 34 kDa 0 281 Elongation factor 1-‐gamma OS=Homo sapiens GN=EEF1G PE=1 SV=3 EF1G_HUMAN (+12) 50 kDa 2 082 Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4 EF2_HUMAN (+7) 95 kDa 0 083 Importin-‐8 OS=Homo sapiens GN=IPO8 PE=1 SV=2 IPO8_HUMAN (+2) 120 kDa 2 084 26S proteasome non-‐ATPase regulatory subunit 2 OS=Homo sapiens GN=PSMD2 PE=1 SV=3 PSMD2_HUMAN (+25) 100 kDa 0 285 Tumor rejection antigen (Gp96) 1 OS=Homo sapiens GN=TRA1 PE=2 SV=1 Q5CAQ5_HUMAN (+14) 92 kDa 2 086 Methionyl-‐tRNA synthetase, cytoplasmic OS=Homo sapiens GN=MARS PE=1 SV=2 SYMC_HUMAN (+4) 101 kDa 2 087 Myosin phosphatase Rho interacting protein OS=Homo sapiens GN=MPRIP PE=2 SV=1 B9EGI2_HUMAN (+1) 118 kDa 0 288 Arginyl-‐tRNA synthetase, cytoplasmic OS=Homo sapiens GN=RARS PE=1 SV=2 SYRC_HUMAN (+1) 75 kDa 2 089 Methylosome subunit pICln OS=Homo sapiens GN=CLNS1A PE=1 SV=1 ICLN_HUMAN (+1) 26 kDa 0 190 Topoisomerase (DNA) I OS=Homo sapiens GN=TOP1 PE=2 SV=1 B9EG90_HUMAN (+10) 91 kDa 1 091 Caprin-‐1 OS=Homo sapiens GN=CAPRIN1 PE=1 SV=2 CAPR1_HUMAN (+9) 78 kDa 1 092 Exportin-‐2 OS=Homo sapiens GN=CSE1L PE=1 SV=3 XPO2_HUMAN (+15) 110 kDa 1 093 Cooperator of PRMT5 OS=Homo sapiens GN=COPR5 PE=1 SV=3 COPR5_HUMAN 20 kDa 1 094 39S ribosomal protein L19, mitochondrial OS=Homo sapiens GN=MRPL19 PE=1 SV=2 RM19_HUMAN (+1) 34 kDa 1 0
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