S2

Supplementary Figure S1. Effects of different functionalized nanomaterials with

similar carboxylated surface on stabilization of i-motif structure.

UV melting profiles of i-motif in the presence of carboxylated grapheme(a), carbon

dots(b) ,CdTe QDs(c) and SWNTs (d) at 2μg/ml, 5μg/ml and 10μg/ml, respectively, in

pH7.0 cacodylic buffer. DNA concentration was 1μM for i-motif in base. Normalized

absorption changes at 260 nm for i-motif were plotted against temperature. As shown

in this Figure, only carboxyl-modified SWNTs selectively induced the stabilization of

human telomeric i-motif, carboxylated graphene, carbon dots and CdTe QDs did not

show this effect. In our previous works, carboxylate-modified gold-nanoparticles also

did not exhibit this effect. Therefore, the selective induction of i-motif formation is

specific for carboxylated SWNTs, other nanomaterials displaying the same functional

surface did not possess this ability.

db

10 20 30 40 50 60 70

0.0

0.2

0.4

0.6

0.8

1.0

N

orm

aliz

ed A

260

nm

Temperature (℃)

1 μM i-motif + 2 μg/mL GO-COOH + 5 μg/mL GO-COOH + 10 μg/mL GO-COOH

GO-COOH

20 30 40 50 60 70

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

A 2

60nm

Temperature (℃)

1 μM i-motif + 2 μg/mL QDs + 5 μg/mL QDs + 10 μg/mL QDs

QDs

20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

A 2

60nm

Temperature (℃)

1 μM i-motif + 2 μg/mL SWNT-COOH + 5 μg/mL SWNT-COOH + 10 μg/mL SWNT-COOH

SWNT-COOH CDs

a c

10 20 30 40 50 60 70

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

A 2

60nm

Temperature (℃)

1μM i-motif + 2 μg/mL CDs + 5 μg/mLCDs + 10 μg/mL CDs

S3

Supplementary Figure S2. i-motif formation induced by SWNTs from the

telomeric dsDNA.

Native PAGE images of G-DNA/C-DNA in the absence or presence of the

SWNT-COOH at pH 7.0. Lane 1, G-DNA; lane 2, C-DNA; lane 3, 1:1 mixture of

G-DNA/C-DNA; lane 4-7, 1:1 mixture of G-DNA/C-DNA in the presence of 2, 4, 8,

16 μg/ml SWNT-COOH, respectively. Twenty percent PAGE and TB running buffer

were used in the experiments at room temperature. The gels were silver-stained. As

shown in this figure and our previous results11,13-17, the telomeric duplex dissociation

and i-motif formation induced by SWNT-COOH was dose-dependent, and at the

concentration of 16 μg/ml SWNT-COOH, approximately 25% telomeric duplex DNA

convert into i-motif structures quantitated using Lab works 4.5 image analysis

software.

G-D

NA

/C-D

NA

G-D

NA

C-D

NA G-DNA/C-DNA + SWNTs

2 4 8 16 (μg/ml)G-D

NA

/C-D

NA

G-D

NA

C-D

NA G-DNA/C-DNA + SWNTs

2 4 8 16 (μg/ml)G-D

NA

C-D

NA G-DNA/C-DNA + SWNTs

2 4 8 16 (μg/ml)

S4

Supplementary Figure S3. Extension of telomeric ssDNA and/or dsDNA, or

TS-oligo by telomerase in the presence or absence of SWNTs.

(a) Extension of C-DNA/G-DNA duplex, G-DNA and C-DNA by telomerase using

the TRAP-G4 method in the presence or absence of SWNTs (0.5μg/ml). The left panel

shows a ladder of amplified extension products stained with 0.2% AgNO3 after gel

electrophoresis; IS indicates the bands of internal standard. Right, Telomerase activity

was quantitated as the percent of the corresponding control sample. The mean of three

independent experiments with comparable results is shown. Error bars indicate ± SD,

n=3. ＊P<0.05, ＊＊P<0.01, one-tailed student′s t-test. (b) Extension of TS-oligo by

telomerase in TRAP assay in the absence or presence of SWNTs (0.5μg/ml, 2 μg/ml

and 8μg/ml, respectively). The left panel shows a ladder of amplified extension

products stained with 0.2% AgNO3 after gel electrophoresis; IS indicates the bands of

internal standard. Right, Telomerase activity was quantitated as the percent of the

corresponding control sample. The mean of three independent experiments with

comparable results is shown. Error bars indicate ± SD, n=3.

b

IS

0 0.5 2 8

SWNT-COOH (μg/ml)

0 0.5 2 80

20

40

60

80

100

% T

elom

eras

e ac

tivity

SWNT-COOH (μg/ml)

G-D

NA

/C-D

NA

G-D

NA

G-D

NA

+SW

NT

C-D

NA

C-D

NA

+ SW

NT

G-D

NA

/C-D

NA

+SW

NT

IS

G-D

NA

/C-D

NA

G-D

NA

G-D

NA

+SW

NT

C-D

NA

C-D

NA

+ SW

NT

G-D

NA

/C-D

NA

+SW

NT

ISIS0

20

40

60

80

100

% T

elom

eras

e ac

tivity

G-DNA/C-DNA G-DNA/C-DNA+SWNT-COOH G-DNA G-DNA+SWNT-COOH C-DNA C-DNA+SWNT-COOH

＊

＊＊ ＊＊ ＊＊＊＊

0

20

40

60

80

100

% T

elom

eras

e ac

tivity

G-DNA/C-DNA G-DNA/C-DNA+SWNT-COOH G-DNA G-DNA+SWNT-COOH C-DNA C-DNA+SWNT-COOH

＊

＊＊ ＊＊ ＊＊＊＊

a

S5

Supplementary Figure S4. Characterization of SWNT-COOH and

MWNT-COOH used in this study with SEM and TEM.

SEM and TEM images of MWNT-COOH (scale bar=100nm, 10nm, respectively, left)

and SWNT-COOH (scale bar=100nm, 2nm, respectively, right).

MWNT-COOH SWNT-COOH

100nm 10nm 100nm 2nm

S6

Supplementary Figure S5. SWNT-COOH was modified with PEG and FITC.

(a) Scheme for modification of SWNT-COOH with PEG followed by FITC. (b) AFM

images of SWNT-COOH and modified with PEG.

SWNT

5 nm

SWNT-PEG

5 nm

a

b

S7

Supplementary Figure S6. UV, FL and IR characterization of synthesized

PEG-functionalized SWNT-COOH.

(a) UV-Vis absorption spectra of SWNT, SWNT-PEG, SWNT-PEG-FITC and FITC.

(b) Fluorescence spectra of SWNT, SWNT-PEG and SWNT-PEG-FITC. (c) Near-IR

absorption spectra of SWNT and SWNT-PEG.

IRc

UV-Vis a

FL b

200 300 400 500 600 700 800-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Abs

Wavelength / nm

SWNT SWNT-PEG SWNT-PEG-FITC FITC

500 550 600 650 700

0

100

200

300

400

FL In

tens

ity (a

.u.)

Wavelength / nm

SWNT SWNT-PEG SWNT-PEG-FITC

0 1000 2000 3000 4000

T

rans

mitt

ance

Wavenumber(cm-1)

SWNT SWNT-PEG

1730

1635

1543

28602920

S8

Supplementary Figure S7. SWNTs were uptaken by cells through lysosome

pathway and in an energy dependent manner.

(a) SWNT-PEG-FITC localizes within the lysosomes after 30min incubation with

Hela cells. The lysosome was stained with Lysotracker and the nuclear was visualized

with DAPI, Merged signal was shown on the right. (b) Hela cells can untake

SWNT-PEG-FITC after incubation for 2-3h at 37 , they cannot take up ℃

SWNT-PEG-FITC efficiently at 4 . ℃

SWNT-PEG-FITC Lysotracker DAPI Merge a

37℃

4℃

SWNT-PEG-FITC DAPI Mergeb

S9

Supplementary Figure S8. Time course of telomerase inhibition induced by

SWNT-COOH in K562 and Hela cells.

Time course of telomerase inhibition induced by carboxylated SWNTs at 25 μg/ml in

K562 and Hela cells. Cells were treated with carboxylated SWNTs (25 μg/ml) for 3, 6,

9, and 12 days, respectively. CHAPS extract was prepared at indicated timepoints and

equivalent amounts of protein (500 ng) were subjected to a standard TRAP assay.

Enzyme activity is represented as the percentage of untreated sample at time zero. The

mean of three independent experiments with comparable results is shown. Error bars

indicate ± SD, n=3.＊P<0.05, ＊＊P<0.01, one-tailed t-test.

0 3 6 9 120

20

40

60

80

100

** ** ** ***

*

*

*

*

% T

elom

eras

e A

ctiv

ity

Days

K562 Hela

*

S10

Supplementary Figure S9. Effects of SWNT-COOH on hTERT cellular

localization and its expression in Hela cells. (a) SWNT-COOH treatment did not

induce the change of localization of hTERT in Hela cells. After treatment with

SWNT-COOH for 1 week, Hela cells were stained with anti-hTERT antibody (red),

and the nucleus was visualized with DAPI staining (blue), and the merged image was

shown in the right. (b) SWNT-COOH did not induce the change of expression of

hTERT, hTEP1 and hTR at mRNA levels by using semi-quantitative RT-PCR assay.

β2-M was used as loading control. (c) SWNT-COOH did not induce the alternation of

expression of hTERT and hTEP1 at protein levels. After one week of treatment, the

protein was extracted and subjected to Western-blotting, then probed with

anti-hTERT and anti-hTEP1 antibodies. β-actin was used as loading control.

a

SWNT-COOH (50μgml)

Control

Merge hTERT DAPI

b

β2-M

hTR

hTEP1

hTERT

(μg/ml) 0 5 10 25 50 c

β-actin

hTEP1

hTERT

(μg/ml) 0 5 10 25 50

S11

Supplementary Figure S10. Acute cytotoxicity induced by carboxylated SWNTs

in K562 and Hela cells. (a) Cell viability of K562 and Hela cells treated with

carboxylated SWNTs after a short-term. The cell viability was determined by Trypan

blue exclusion at a seeding concentration of 1×104/well in a 24-well plate, and cell

numbers were counted at day 3 and 6, respectively. (b) About 2×103 K562 and Hela

cells were seeded in 96-well plates and incubated at 37℃ in the presence of SWNTs

with increasing concentrations (1-100 μg/ml) for 3 and 6 days. Cell viability was

determined by XTT assay and the absorbance at 450 nm was recorded. The values are

obtained from three experiments and expressed as means ± SD, n=3.

0 5 25 50 1000

10

20

30

40

50C

ell N

umbe

rs (×

104 )

SWNT-COOH (μg/ml)

3 days 6 days

0 5 25 50 1000

2

4

6

8

10

12

Cel

l Num

ber

(×10

4 )

SWNT-COOH (μg/ml)

3 days 6 days

a

0 1 5 10 25 50 1000.0

0.5

1.0

1.5

OD

450

SWNT-COOH (μg/ml)

3 days 6 days

K562

0 1 5 10 25 50 1000.0

0.4

0.8

1.2O

D 4

50

SWNT-COOH (μg/ml)

3 days 6 days

Hela

b

K562 Hela

S12

Supplementary Figure S11. Telomerase activity inhibition induced by

transfection of 2′-O-MeRNA and expressing TRF2△B△M

(a) Telomerase activity inhibition induced by transfection of 2′-O-MeRNA ranging

from 0.02μM to 2μM for 72h in Hela cells (Left). Telomerase activity was quantitated

as the percent of the corresponding control sample (Right). (b) Telomerase activity

inhibition induced by transfection of TRF2△B△M for 3d, 6d, 9d and 12d, respectively,

in Hela cells (Left). Telomerase activity was quantitated as the percent of the

corresponding control sample (Right). The values are obtained from three experiments

and expressed as means ± SD, n=3.

a 0.02μM 2μMCtrl.

2′-O-MeRNA

IS

0.02μM 2μMCtrl.

2′-O-MeRNA

IS

b TRF2△B△M

3d 6d 9d 12dCtrl.

IS

TRF2△B△M

3d 6d 9d 12dCtrl.TRF2△B△M

3d 6d 9d 12dCtrl.

IS

0 0.02 0.07 0.2 0.7 20

20

40

60

80

100

% T

elom

eras

e ac

tivity

2' -O-MeRNA (μM)

Ctrl. 3d 6d 9d 12d0

20

40

60

80

100

% T

elom

eras

e ac

tivity

TRF2ΔBΔM

S13

Supplementary Figure S12. Identification of stably expression of TRF2ΔBΔM in

K562 and Hela cells.

The expression of TRF2ΔBΔM in pCDNA3.1-myc-TRF2ΔBΔM-transfected and empty

vector-transfected K562 and Hela cells, which was identified by the anti-myc-tag

antibody. β-actin was used as a loading control.

Myc-tag

β-actin

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

HeLa

β-actin

Myc-tag

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

K562

Myc-tag

β-actin

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

HeLa

Myc-tag

β-actin

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

HeLa

β-actin

Myc-tag

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

K562

β-actin

Myc-tag

pCD

NA

3.1-

myc

pCD

NA

3.1-

myc

-TR

F2△

B△M

K562

S14

Supplementary Figure S13. Cellular uptake of low level of SWNTs in long-term

exposed Hela cells. Hela cells were continuously grown in the presence or absence of

10μg/ml SWNT for 20d and 40d, respectively. (a) After washing the cells to remove

free SWNT, the pellet of long-term treated Hela cells appears completely black

compared with untreated cells. (b) Fluorescence for SWNT-PEG-FITC in Hela cells

after incubation for 20d and 40d, respectively. (c) Fluorescence for SWNT-PEG-FITC

in Hela cells after continuous treatment for 40d followed by cultured in

SWNT-PEG-FITC-free medium for 3 days, or 6 days, respectively. Evident loss of

SWNTs from cells was observed.

Control SWNTs (20d)

SWNTs (40d)

a

Control SWNTs (20d) SWNTs(40d) b

SWNTs(40d) →free medium for 3 days SWNTs(40d) →free medium for 6 days c

S15

Supplementary Figure S14. Cell proliferation suppression and telomere loss

induced by long-term exposure of Hela cells to carboxylated SWNTs.

(a) Cell proliferation defects of long-term exposure of Hela cells to carboxylated

SWNTs. Hela cells were plated at 1.25×105 cells/flask, incubated in the presence or

absence of varied concentrations of SWNTs (1 μg/ml, 5 μg/ml and 10 μg/ml,

respectively) for 8 weeks, the cells were numbered and passaged weekly, and

reseeded with 1.25×105 cells until cell numbers were too low to reseed. At 42 days,

cells treated with 10 μg/ml were cultured in SWNTs-free medium for further 2 weeks.

The proliferation is expressed in population doublings as a function of days of culture.

This data is a representative of three experiments. For positive control, growth curve

for transfection of 2′-O-MeRNA in Hela cells was also performed. (b) Hybridization

protection assay (HPA) was performed on genomic DNA isolated from Hela cells

treated with either 2′-O-MeRNA or SWNT-COOH (10μg/ml) for 56 days to assess

the length of G overhang and total telomere length. ExoI nuclease digestion was used

to assess integrity of the 3′ overhang. At 42 days, cells treated with 10 μg/ml were

cultured in SWNT-free medium for further 2 weeks, and the G overhang and total

telomere length was also determined. Luminescence intensity in arbitrary units (AU)

was normalized against Alu probe. The mean of three independent experiments with

comparable results is shown. Error bars indicate ±SD, n=3.**P<0.01, two-tailed

student′s t-test.

a b

0 7 14 21 28 35 42 49 56

0

10

20

30

40

50

60

PDs

Days

Control SWNT(1μg/ml) SWNT(5μg/ml) SWNT(10μg/ml) SWNT(10μg/ml)→SWNT-free medium 2' -O-MeRNA

0

2

4

6

8

10

12

**** **

******

****

total telomere+Exo1

total telomereG-overhang+Exo1

G-overhangLum

ines

cenc

e(a.

u.:t

elom

ere/

Alu

) Control SWNT-COOH (10μg/ml)

SWNT-COOH (10μg/ml)→SWNT-free medium 2' -O-MeRNA

**

S16

Supplementary Figure S15. Localizations of PCBP1, TRF2 and TRF1 at

telomere. (a) Colocalization of PCBP1 with TRF1 and TRF2 in Hela cells. Images

are shown of DAPI stain (nuclei), PCBP1 immunostaining (green), TRF1 or TRF2

immunostaining (red), and merged PCBP1 plus TRF1 or PCBP1 plus TRF2 images.

Yellow granules in the merged images indicate regions of colocalization. (b)

Quantitation of colocalization of PCBP1/TRF1 and PCBP1/TRF2 was carried out

using Image J software (n=25 cells). (c) Telo-FISH of PCBP1 colocalization with

telomeric probe. Subconfluent proliferating Hela cells on slides were fixed and

hybridized to a Cy3-labeled telomere-specific PNA probe. The slides were

immunostained with PCBP1, TRF1, or TRF2 primary antibody and secondary

antibody conjugated to fluorescein or DyLightTM488 and processed for confocal

imaging. Images are shown of DAPI stain (blue), PNA probe (red), PCBP1 (green),

TRF1 (green), or TRF2 (green). (d) Quantification of colocalization of PCBP1, TRF1,

or TRF2 with the PNA probe (yellow granules in merged images) was carried out

using Image J software (n=28) or by manual counting of the percentage of total

telomeric granules (yellow+red) that is colocalized (yellow) with PCBP1, TRF1, or

TRF2 (n=3 cells).

PCBP1 TRF1 Merge

PCBP1 TRF2 Merge

PCBP1 Telo-FISH Merge

TRF1 Telo-FISH Merge

TRF2 Telo-FISH Merge

0

20

40

60

80

100

PCBP1/TRF2PCBP1/TRF1% C

ells

(﹥4

colo

caliz

atio

ns)

0

20

40

60

80

100

TRF2/Telo-FISH

TRF1/Telo-FISH

% C

ells

(﹥4

colo

caliz

atio

ns)

PCBP1/Telo-FISH

a

b

c

d

S17

Supplementary Figure S16. PCBP1 associates with TRF1 and TRF2 in vivo in

Hela cells. Subconfluent proliferating Hela cells were harvested and subjected to

reciprocal IP-Western blotting for PCBP1/TRF1 (a) or for PCBP1/TRF2 (b). Control

IPs were carried out using an equal quantity of the same type of non-immune IgG.

ba

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: PCBP1

IB: TRF1

IB: PCBP1

IB: TRF2

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: TRF1

IB: PCBP1

Con

trol

IPIn

put (

10%

)IP

: TR

F1

IB: TRF2

IB: PCBP1

Con

trol

IPIn

put (

10%

)IP

: TR

F2

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: PCBP1

IB: TRF1

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: PCBP1

IB: TRF1

IB: PCBP1

IB: TRF2

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: PCBP1

IB: TRF2

Con

trol

IPIn

put (

10%

)IP

: PC

BP1

IB: TRF1

IB: PCBP1

Con

trol

IPIn

put (

10%

)IP

: TR

F1

IB: TRF2

IB: PCBP1

Con

trol

IPIn

put (

10%

)IP

: TR

F2

IB: TRF2

IB: PCBP1

Con

trol

IPIn

put (

10%

)IP

: TR

F2

S18

Supplementary Table S1. Physicochemical characterization of different

functionalized CNTs.

Types Size distribution

(nm)

Average diameter

(nm)

Zeta potential

(mV in cellular media)

SWNT-COOH 180.56±3.25 1.1±0.25 -25.45±2.45

SWNT-CH2OH 178.2±1.85 1.15±0.15 -15.32±1.14

SWNT-CONHCH2CH2NH2 181.24±2.75 1.21±0.27 16.56±2.34

SWNT-PEG 186.2±3.24 2.65±0.35 -28.37±3.24

MWNT-COOH 205.25±2.15 16.45±1.34 -35.25±2.56

The size distribution, surface coating, surface charges were characterized using

differential techniques described in the Supplementary Methods. (data represent mean

±SD, n=3)

S19

Supplementary Table S2. IC50 values of different functionalized SWNTs and MWNTs on telomerase activity inhibition in K562 and Hela cells.

aIC50 values obtained using standard TRAP assay after 6 days of treatment. Results are means of triplicates (±SD) ND, not determined, since there was no decrease in telomerase activity after treatment with indicated CNTs doses.

IC50 values of different functionalized SWNTs and MWNTs for telomerase activity

inhibition in K562 and Hela cells. Cells were treated with increasing concentrations of

different functionalized CNTs (1-100 μg/ml) for 6 days, CHAPS extract was prepared

and equivalent amounts of protein (500 ng) were subjected to a standard TRAP assay.

Telomerase activity was quantitated as the percent of the corresponding control

sample containing no CNTs.

IC50 (μg/ml)a CNT type K562 Hela

SWNT-COOH 10.2±0.8 7.5±0.45 SWNT-CH2OH 47.2±1.53 36.8±1.24 SWNT-CONHCH2CH2NH2 ND ND MWNT-COOH ND ND

S20

Supplementary Methods

Preparation of PEG-functionalized SWNTs

The carboxylated SWNT sample was refluxed in thionyl chloride for 12 h, followed

by a complete removal of unreacted thionyl chloride on a rotary evaporator with a

vacuum pump. The PEG diamine with ～35 repeating units and a molecular weight

of 1500 (PEG1500N; Fluka, USA) was added to the treated nanotube sample and the

mixture was heated to 120 °C and stirred for 3 days under nitrogen protection. The

reaction mixture was cooled to ambient temperature and then extracted repeatedly

with water. The soluble fraction containing the PEG1500N-functionalized SWNTs was

separated from the insoluble residue via centrifuging at ~1400 ×g for 15 min. The

excess free PEG1500N was removed via dialysis (tubing MWCO~12,000) against fresh

deionized water for 3 days to obtain the PEG1500N-SWNT sample27 (Supplementary

Ref. 61).

The unused primary amine groups in PEG1500N-SWNTs were targeted in the labeling

with the fluorescent dye FITC (fluorescein isothocyanate). In the experiment, a

freshly prepared solution of FITC in dimethyl sulfoxide (1 mg/mL) was mixed with

an aqueous PEG1500N-SWNT solution (pH=9). The reaction was carried out in the

dark at 4 °C for 12 h. The reaction mixture was dialyzed against fresh deionized water

for 3 days to obtain the FITC-labeled PEG1500N-SWNT sample27. All of the samples

were readily soluble in water, and the resulting aqueous solutions remained stable

without any precipitation under ambient condition (at least within the observation

period of several months).

Preparation of carboxyl-modified graphene, carbon dots and CdTe QDs.

Carboxylmodified graphene (GO-COOH) was prepared according to the method

reported by Sun et al and our previous works (Supplementary Ref. 62-64). The stock

solution of GO-COOH (1mg/mL) was obtained by sonicating the final product for 2 h

in pH 7.0 aqueous solution.

S21

Carbon dots (CDs) were prepared from candle soot as our reported method

(Supplementary Ref. 65-66). Candle soot was refluxed in 5 M nitric acid for 12 h, and

purified by centrifugation at 13000 rpm for 30 min to remove unreacted soot and

non-fluorescent large sized particles. The obtained supernatant CDs were neutralized

by Na2CO3 and dialyzed against pure water.

The water-soluble CdTe QDs were synthesized based on previous publication

(Supplementary Ref. 67). Tellurium powder was used to prepare the NaHTe aqueous

solution. In brief, it was reduced by excessive NaBH4 in water under stirring and N2

bubbling. After the Te was completely reduced, a certain volume of the NaHTe

solution was injected into CdCl2-MPA ( N,N-methylenebisacrylamide ) solution,

which was deaerated by N2 for 20 min. The molar ratio of Cd2+/HTe-/MPA was set as

1: 0.5: 2.4. And then, it was heated until boiling. Under refluxing, fluorescence of the

solution appeared and could be tuned in color by prolonging the refluxing time.

Measurement

SEM images of SWNTs and MWNTs were obtained on a HITACHI S-4800 scanning

electron microscope. Samples were prepared by pipetting 5 μL of colloid solution

onto a silicon substrate pretreated with piranha etch solution (4:1 concentrated

H2SO4/30%H2O2) for 1 h at room temperature. After evaporation of the solvent, the

substrate was dried overnight under vacuum.

Transmission electron microscopic (TEM) experiments were performed using a

Philips Tacnai G2 20 S-TWIN microscope operating at 200 kV. For visualization by

TEM, samples were prepared by dropping a solution of production on a copper grid.

Atomic-force microscopy (AFM) measurements were performed using a Nanoscope

V multimode atomic force microscope (Veeco Instruments, USA) under ambient

conditions, and samples were prepared by dropping the solution on mica.

Fluorescence measurements were carried out using a JASCO FP-6500

spectrofluorometer with the slit width for the excitation and emission of 5 nm. FTIR

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characterization was carried out on a BRUKE Vertex 70 FTIR spectrometer. UV-vis

absorbance measurements was carried out on a Jasco-V550 UV/Vis

spectrophotometer. The dynamic light scattering (DLS) measurements and the zeta

potential of CNTs in DMEM media (~0.05 mg/mL) was determined using a Zeta

PALS, zeta potential analyzer (Brookhaven Instruments Corp. Holtsville, NY).

UV melting experiment

Absorbance measurements and melting experiments were carried out by using a Cary

300 UV/Vis spectrophotometer equipped with a Peltier temperature control accessory.

All UV/Vis spectra were measured in a 1.0 cm path-length cell with the same

concentration of cosolutes and SWNT aqueous solution accordingly as the reference

solution. Absorbance changes at λ=260 nm versus temperature were collected at a

heating rate of 1℃min-1. The melting measurements for each sample were repeated at

least three times. Primary data were transferred to the graphics program Origin for

plotting and analysis.

Native PAGE

Native PAGE experiments were carried out in 0.045 M Tris-borate buffer. G-DNA

/C-DNA duplex was formed by mixing an equimolar concentration of G-quadruplex

and i-motif for 12 h at 4°C in 0.1 mM sodium cacodylate and 100 mM NaCl buffer

(pH 7.0). Then, SWNT-COOH was added into the duplex at a 1 μg/ml: 2μM ratio and

incubated for 12 h at 4°C. Electrophoresis was carried out by using 20% acrylamide at

200 V for 50 min at room temperature. The gels were silver-stained.

Bioassays

Semi-quantitative RT-PCR Analysis

Two step RT-PCR was performed using the TaKaRa RNA PCR kit (AMV) Ver.3.0

following manufacturer’s protocol. The following primers were used: hTERT,

forward primer (5’-CGTGGTTTCTGTGTGGTGTC-3’) and reverse primer

(5’-CCTTGTCGCCTGAGGAGTAG-3’); hTEP1, forward primer

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(5′-AGCAGAAGAGCCACTGAAAGCA-3′) and reverse primer

(5′-CTTCGCTGTCTGAGGAAATCAG-3′); hTR, forward primer

(5’-GCCTTCCACCGTTCATTCTA-3’) and reverse primer

(5’-GCTGACAGAGCCCAACTCTT-3’), β2-M, forward primer

(5’-GAGAGACCCTCACTGCTG-3’) and reverse primer

(5’-GATGGTACATGACAAGGTGC-3’). PCR products were run on 2% agarose gel

and viewed under UVP gel documentation system (Ultraviolet Products, Upland, CA,

USA). Quantifications were performed using Lab works 4.5 image analysis software.

Immunofluorescence

Immunofluorescence was performed as previously reported18.37-38. Cells were fixed in

2% formaldehyde and permeabilized in 0.25% Triton X100 in PBS for 5 min at room

temperature. For immunolabeling, cells were incubated with primary antibody and

then washed in PBS and incubated with the fluorophore-conjugated secondary

antibodies. The following primary antibodies were used: mAb anti-TRF1 (Novus),

mAb anti-TRF2 (Novus), pAb anti-POT1 (Sigma), mAb anti-γ-H2AX (Genscript),

mAb anti-53BP1 (Novus), mAb anti-hTERT (Rockland), mAb anti-PCBP1

(Proteintech). The following secondary antibodies were used: Rhodamine or

DyLightTM488 conjugated goat anti-rabbit, fluorescein or DyLightTM594 conjugated

goat anti-mouse (Jackson Laboratory). Fluorescence signals were captured by using

Olympus Fluoview FV1000 confocal microscope and analyzed by FV10-ASW 1.6

Viewer program (Olympus, Japan).

Immunoblotting analysis

Cells after treatment were washed with PBS, and lysed in 100 μl of lysis buffer (10

mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 25 mM NaF, fresh 100 mM

Na3VO4 and l mM dithiothreitol). Cell lysates were centrifuged for 10 min at 12000g.

Concentrations of protein in the supernatant were determined by Bradford protein

assay. Equal amounts of protein (40 μg) were resolved on 10% SDS-PAGE, and

transferred electrophoretically to PVDF membrane. The membranes were blocked

with nonfat dry milk (5%) in PBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05%

S24

Tween-20), and then incubated with primary antibodies for 2 h at room temperature,

washed three times with PBST, and then incubated with secondary antibody

(HRP-conjugated) for 2h at room temperature. After washing the secondary antibody,

the bound antibody complex was detected using an ECL chemiluminescence reagent

(Thermo). The primary antibodies used in this experiment were: mAb anti-TRF1

(Novus), mAb anti-TRF2 (Novus), pAb anti-POT1 (Sigma), mAb anti-γ-H2AX

(Genscript), pAb anti-H2AX (Genscript), mAb anti-hTERT (Rockland), pAb

anti-hTEP1(Santa Cruz), mAb anti-PCBP1 (Proteintech), anti-p21 and anti-p16

(Thermo Fisher Scientific), β-actin (Sigma), and anti-myc-tag (Sungene Biotech).

Short-Term Cell Viability Assay

Cell viability was measured by XTT assay18,30. Cells were seeded on 96-well plates

(2.0×103/well) and pre-incubated for 24 h at 37°C in a humidified atmosphere of 5%

CO2 before exposure to different dilutions of carboxylated SWNTs for 3 and 6 days.

After washing with PBS, 50 μl of XTT reagents (Cell Proliferation kit, KeyGEN) was

added to each well, the cells were incubated for 3 h at 37°C, and absorbance (450 nm)

was measured to estimate cell viability on a Bio-Rad model-680 microplate reader.

The cell viability was also determined by Trypan blue exclusion at a seeding

concentration of 1×104/well in a 24-well plate, and cell numbers were counted on day

3 and 6, respectively.

Cytogenetic Analysis

To determine the presence of anaphase bridges, cells were seeded on glass coverslips

in complete culture medium and treated with carboxylated SWNTs or transfected with

2′-O-MeRNA, TRF2△B△M for one week, then stained with DAPI (Sigma) and

mounted. Images of anaphases were recorded with an Olympus BX-51 fluorescence

microscope (Tokyo, Japan) coupled with a CCD camera controlled by DP 70 software.

The frequency of anaphase bridges/micronuclei was calculated as the ratio between

cells exhibiting anaphase bridges/micronuclei and the total number of anaphase cells.

At least 50 anaphase cells were examined in each experiment.

Chromosome aberrations were evaluated as previously reported37-39. To obtain

chromosome preparations, cells in the log phase of growth were incubated with

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0.1μg/ml colchicine for 2 h and trypsinized, then incubated with hypotonic 0.075 M

KCl for 10 min, fixed with methanol/acetic acid (3:1, v/v), dropped onto frosted

microscope slides, and air-dried overnight. Chromosomal aberrations were blindly

evaluated by two independent observers in Giemsa- and DAPI-stained metaphases

from two grown cultures for each treatment. Analysis was performed at day 7 of

treatment.

Cell Cycle Analysis

Briefly, after two weeks treatment with carboxylated SWNTs or transfection with

2′-O-MeRNA or TRF2△B△M, cells (0.5-1×106) were washed with PBS, fixed in 70%

ethanol and kept at -20℃ for at least 24 h. They were then washed in PBS and

resuspended in 50 μg/ml propidium iodide and 100 μg/ml RNase in PBS18,60. The cell

suspension was incubated for 30 min at room temperature in dark and cell cycle

distribution was determined by flow cytometry (FACSCalibur, Becton-Dickinson),

with CellQuest software. The sub-G1 peak was also quantified as apoptotic cells18.

Apoptosis analysis

Surface exposure of phosphatidylserine in apoptotic cells was measured using an

AnnexinV/FITC and PI apoptosis detection kit (KeyGEN, Nanjing, China)18. The

cells were collected and resuspended in the binding buffer provided in the kit, then

mixed with PI and FITC conjugated Annexin V. After incubation for 15 min, the cells

were assessed via flow cytometric analysis.

SA-β-Gal assay

The senescent cells were verified by staining for SA-β-Gal as described previously 30.

Cells treated with carboxylated SWNTs or transfection with 2′-O-MeRNA or

TRF2△B△M were washed twice in PBS, fixed in 2% formaldehyde/0.2%

glutaraldehyde for 5 min at room temperature, washed again in PBS, and incubated

for 16h with β-Gal stain solution containing 1 mg/ml

5-bromo-4-chloro-3-indolyl-β-D-galactoside, 40 mM citric acid/sodium phosphate

(pH 6), 5 mM potassium ferrocynide, 5 mM ferricyanide, 150 nM NaCl and 2 mM

MgCl2. Cells were viewed with a OLYMPUS BX-51 light microscope and

photographed.

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Telo-FISH Assays

Combined telomere DNA staining (FISH probe) and immunostaining (PCBP1,TRF1

or TRF2) was performed as described before37-38,41. Cells cultured in 6-well culture

slide chambers were fixed for 8 min at 37°C with 2.0% paraformaldehyde and rinsed

three times with PBS. Fixed samples were placed in deionized water followed by

deionized water plus 0.2% Tween 20. Samples then underwent antigen retrieval in

citrate buffer, followed by a 5-min 95% ethanol treatment and air drying. Samples

were hybridized with a Cy3-labeled telomere-specific peptide nucleic acid (PNA)

probe with nucleotide sequence 5′-CCCTAACCCTAACCCTAA-3′ (TaKaRa)

(0.3μg/ml PNA in 70% formamide, 10 mM Tris, pH 7.5, 0.5% blocking reagent).

Slides were washed twice in PNA wash solution (70% formamide, 10 mM Tris, pH

7.5, 0.1% albumin), washed three times in PBS plus Tween 20, blocked with 10% calf

serum in PBS at 25°C for 90 min, incubated overnight with primary antibody at 4 °C,

blocked at 37 °C for 20 min, rinsed, and incubated with secondary antibody

conjugated to fluorescein. Cells were then washed twice with PBS, and coverslips

were applied as above. The primary and secondary antibodies were the same as above.

Three independent experiments were performed per assay type per cell line.

Immunuprecipitation

Subconfluent proliferating cells in 150-cm2 dishes were harvested, and whole cell

extracts were prepared and subjected to IP as described before35,38,41. Each IP was

performed using 5μg of antibody or antibody combination and 500μg of cell protein.

Precipitated proteins were collected using protein-G beads, washed, eluted in boiling

Laemmli sample buffer, and subjected to Western blotting. The IP antibodies were

PCBP1 (Proteintech), TRF1 (Novus), TRF2 (Novus), and normal (non-immune) IgG

(mouse IgG, or rabbit IgG, Santa Cruz).

Long-Term Exposure Studies

Cells were seeded in growth medium into T80 tissue culture flasks at 1.25×105 cells

per flask and exposed to a nonacute cytotoxic concentration of carboxylated SWNTs

or transfected with 2′-O-MeRNA every 3 to 4 days. Every 7 days, the cells in control

and treated flasks were trypsinized and counted using a hematocytometer, and flasks

S27

were reseeded with 1.25×105 cells per flask. Remaining cells were collected and used

for measurements. This weekly process, with twice-weekly CNTs addition, was

continued until such time that there were less than 1.25×105 cells available for

reseeding. The cumulated population doubling was measured30.

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