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: john.diffley@cancer.org.uk

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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