Supplemental Materials and Methods

Reagents, cell lines and cell culture

Human prostate cancer cell lines PC3 and LNCaP were obtained from the American Type Culture

Collection. These cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum

(Hyclone), 100 U/ml of penicillin and 100 µg/mL streptomycin, and maintained in an incubator with a

humidified atmosphere of 95% air and 5% CO2 at 37 ºC. Nicotinic acid was purchased from Sigma and

AZD2281 from Selleck Chemicals.

Tissue sample preparation for protein extraction

One pair of frozen benign and prostate cancer tissue specimens were used for this study. Samples were

taken from a patient who underwent radical prostatectomy at the City of Hope (COH) National Medical

Center with final diagnosis of prostatitic adenocarcinoma (Gleason score 7). The samples were snap-

frozen shortly after prostatectomy and had been stored at –80 oC at the COH Tumor Bank since collected.

The protocol for using these samples was approved by the COH Cancer Protocol Review and Monitoring

Committee (CPRMC) and Institutional Review Board (IRB). Cancer and normal/BPH tissues were

harvested from cancer and benign tissue blocks, respectively, using biopsy needles (Miltex, Inc., York,

PA). The areas for sampling were circled on the H&E slides by a pathologist and then marked on the

blocks to achieve high tumor content (> 90%). Up to 10 cores (1.5 mm in diameter and 2-5 mm in depth,

50-100 mg in weight) were punched from each frozen tissue block. Tissues were homogenized with 10

volume of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Sodium deoxycholate, 0.1% SDS, 1% NP-40,

pH 8.0) supplied with Complete Protease Inhibitors (Roche) and PMSF (Sigma). Soluble proteins were

collected after centrifugation for Western blot analysis.

Western blot and immunoprecipitation analysis

Western blot was carried out with the following antibodies: NAMPT (Bethyl), SIRT1 (Epitomics),

NNMT (GenWay), acetylated p53 (Cell Signaling), acetylated FOXO1 (Santa Cruz Biotech), acetylated

lysine (Millipore), FOXO3a (Cell Signaling) and GAPDH (Trevigen). FOXO3a immunoprecipitation

was performed as described previously (Chen et al., 2005) using protein A/G-agarose beads (Santa Cruz

Biotech).

Measurement of cell proliferation and apoptosis

Cell proliferation was analyzed using XTT Cell Proliferation Kit (Roche) per manufacture’s instructions.

Apoptosis was measured by TUNEL assay using a tetramethylrhodamine (TMR red) In Situ Cell Death

Detection Kit (Roche) coupled with flow cytometry or photographed under fluorescent microscope as per

manufacture’s instructions. TMR was excited with 530 nm (green) laser for flow cytometric analysis.

Stable gene knockdown using lentiviral vectors

Short hairpin RNA (shRNA) was designed according to rules described by Reynolds et al (Reynolds et

al., 2004). The sequence of shRNA against NAMPT was 5’-GAGTGTTACTGGCTTACAA-3’, and a

second shRNA (5’-GAGTGTTACGGGGTTCCAG-3’) was from the literature (Yang et al., 2007).

Scrambled shRNA sequence was created by mutating critical bases on shRNA against NAMPT: 5’-

GAGTGTTACGGGGTTCCAG-3’. The sequence of shRNA against SIRT1 was 5’-

GAAGTGCCTCAGATATTAA-3’. All shRNAs were subcloned into the pSicoR lentiviral

vector(Ventura et al., 2004), in which shRNA is driven by a U6 promoter and a reporter green fluorescent

protein (GFP) expression cassette is driven by an immediate early promoter of cytomegalovirus (CMV).

We produced VSV-G (G protein of vesicular stomatitis virus) pseudotyped shRNA lentiviral vectors

using a four-plasmid transfection system described previously (Yuan et al., 2010). Recombinant

lentiviruses for NAMPT or SIRT1 knockdown were referred to shNAMPT or shSIRT1 respectively.

Lentiviral transductions were conducted at a multiplicity of infection (MOI) of 4. Transduced cells were

typically used for further experiments after 3 days of transductions. In addition, we constructed an

inducible lentiviral vector for SIRT1 knockdown. SIRT1 shRNA oligoes were annealed and cloned into

Age1 and EcoR1 sites of pLKO-Tet-On vector (Wiederschain et al., 2009). Viral particles were produced

as described above. Two days after transduction, cells were treated with puromycin (2 μg/ml) and stable

cell line was established. Cells were then treated with doxycycline (100 μg/ml) to induce shSIRT1

expression and gene knockdown.

RNA analysis

Total RNA was extracted using Trizol (Invitrogn), and treated with RNase free DNase (Roche). First

strand cDNA was synthesized using Superscript III kit (Invitrogen). The cDNA was diluted and 2μl was

used for real time PCR using SYBR Green qPCR kit (Invitrogen). Real time PCR was performed on IQ5

multicolor Real-time PCR system (Bio-Rad). Melting curve was generated at the end of PCR cycles to

check the specificity of the primers. The 18s rRNA gene was used as a control. Primers sequences are

available upon request.

Colony formation assay

A standard two-layer soft agarose culture was used with the bottom layer of 0.6% agarose and the top

layer of 0.35% agarose. Prostate cancer cells were seeded in triplicate at 500 cells/well in 6-well plate,

and colonies were counted after 20 days of incubation. Plates were stained with 0.0055% Crystal Violet

for 1 hour, then rinsed with water and colonies were scored with aid of a microscope.

Measurement of SIRT1 deacetylase activity

Change of NAD+-dependent deacetylase activity in prostate cancer cells upon NAMPT knockdown was

determined using SIRT1 Fluorimetric Drug Discovery kit (Biomol) as per the manufacturer’s

recommendations. Non-NAD+ dependent Histone deacetylase (HDAC) activity was blocked by adding

800 nM trichostatin A (Sigma).

NAMPT and SIRT1 over-expression

For NAMPT over-expression, we extracted total RNA from human WI38 fibroblast cells and synthesized

the first strand cDNA by reverse-transcription. NAMPT coding sequence (CDS) was amplified from

cDNA using a pfu DNA polymerase (Strategene) with the following primers: forward 5’-

CTGTCCTCCGGCCCG-3’; reverse 5’-GCAAAGGGCCCTAAAAAGCTTAC-3’. NAMPT CDS was

cloned into TA cloning vector (Invitrogen) and verified by DNA sequencing. To enhance the expression

of NAMPT, a Kozak sequence (GCCACC) was added right before the start codon of NAMPT CDS, and

the entire NAMPT expression sequence was then placed under a cytomegalovirus (CMV) promoter in a

lentiviral vector. For SIRT1 over-expression, a pBabe retroviral vector carrying a SIRT1 expression

cassette, pYESir2-puro (Vaziri et al., 2001), was obtained from Addgene, and retrovirus was packaged

using Phoenix Ampho cell line (ATCC). SIRT1 over-expressing PC3 and LNCaP cells were selected

using puromycin at 1 or 0.5 µg/ml for 1 week, respectively.

In vitro cell invasion assay

This assay was performed using QCM ECMatrix Cell Invasion Fluorometric Assay Kit (Chemicon) as per

the manufacturer’s instructions. PC3 cells were transduced with lentiviral vectors at MOI of 4. Three

days after transduction, cells were incubated in serum-free media for 1 day, and 2x105 cells were then

seeded in the upper chambers. After 48 hours of incubation, migrated cells in the lower chamber was

measured by the fluorometri assay as instructed.

ROS measurement

ROS level was measured using the dye 5-(and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate

(H2DFFDA; Molecular Probes). Following treatment with FK866 for 72hrs, cells were trypsinized and

re-suspended in phosphate buffered saline and loaded with 5 μM H2DFFDA for 30 min at room

temperature. Cells were then centrifuged and re-suspended in growth medium and incubated for 1hr in

CO2 incubator. After washing the dye, cells were analyzed immediately by flow cytometry with excitation

at 488 nm and emission at 515 nm.

NAD+, Glucose, and ATP assays

Cellular NAD+ concentration was measured using NAD/NADH Assay Kit (Abcam) as per the

manufacturer’s instruction. Glucose was analyzed using Glucose Assay Kit (Sigma). Briefly, PC3 cells

were seeded at a density of 5x104 cells/ml in a 24-well plate in duplicates and were treated with FK866

(10-50nM) or DMSO. Media aliquots were collected at different time points and diluted 1:80 in sterile

water for glucose measurement. ATP was measured using Cell Titer Glo Kit (Promega). The cells on 24-

well plates were equilibrated for 30 minutes at room temperature (RT), detached from the plate with 50 μl

Trypsin, resuspended in 250ul of RPMI 1640 media and counted. An aliquot of 100ul cells were then

mixed with an equal volume of Cell Titer Glo reagent, and the contents were placed on an orbital shaker

to induce cell lysis for 2 minutes, followed by incubation at RT for 10 minutes to stabilize the luminescent

signal before obtaining the reading at an integration time of 0.25-1second per well.

References:

Chen WY, Wang DH, Yen RC, Luo J, Gu W, Baylin SB (2005). Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses. Cell 123: 437-48. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A (2004). Rational siRNA design for RNA interference. Nat Biotechnol 22: 326-30.

Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK et al (2001). hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107: 149-59. Ventura A, Meissner A, Dillon CP, McManus M, Sharp PA, Van Parijs L et al (2004). Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci U S A 101: 10380-5. Wiederschain D, Wee S, Chen L, Loo A, Yang G, Huang A et al (2009). Single-vector inducible lentiviral RNAi system for oncology target validation. Cell Cycle 8: 498-504. Yang H, Yang T, Baur JA, Perez E, Matsui T, Carmona JJ et al (2007). Nutrient-Sensitive Mitochondrial NAD(+) Levels Dictate Cell Survival. Cell 130: 1095-107. Yuan H, Wang Z, Gao C, Chen W, Huang Q, Yee JK et al (2010). BCR-ABL gene expression is required for its mutations in a novel KCL-22 cell culture model for acquired resistance of chronic myelogenous leukemia. J Biol Chem 285: 5085-96.

Supplemental Figure Legend

Figure 1 Expression of NNMT in normal human tissues and cancer cell lines

(A) Expression of NNMT in cancer cell lines using normal human liver lysate as a control. KCL-22

lysate was loaded in both lanes 3 and 4. (B) Comparison of NNMT expression in additional normal

human tissues with cancer cell lines. NB, normal breast; NC, normal colon; NL, normal lung; NPM,

normal peripheral blood mononuclear cells.

Figure 2 Validation of NAMPT antibody specificity

Whole gel images of Western blots using NAMPT antibody. Gel 1: normal human liver lysate (lane 1)

and hepatic cancer cell line HepG2 (lane 5) as positive controls, as compared to CML cell lines (K562

and KCL-22). Note that on lane 4, KCL-22 cell lysate was intentionally overloaded. Gel 2: analysis of

CML cell lines along with mononuclear cells from normal human peripheral blood was done by

simultaneous probing with NAMPT and GAPDH antibodies. Gel 3: analysis of normal prostate, BPH-

bearing prostate tissues and prostate carcinoma lysates. All these samples were obtained from the same

patient. Normal and BPH-bearing tissues were from the same benign tissue block. Although BPH sample

was collected from a BPH area, the purity of BPH sample was uncertain due to the use of biopsy needles

which might punch through the BPH gland and thus collect certain amount of normal tissue underneath.

Such procedure would undermine the detection of moderate change of gene expression in BPH-bearing

tissue. In all gels, however, NAMPT antibody predominantly detected the NAMPT protein.

Figure 3 NAMPT expression in normal and tumor tissues

(A) Normal human tissues with high levels of NAMPT expression. (B) Normal human tissues with low

levels of NAMPT expression, but increased in their corresponding tumors.

Figure 4 Effects of a second set of NAMPT shRNA

NAMPT was knocked down by a second set of shRNA in PC3 cells, which sensitized the cells to

hydrogen peroxide treatment. The assay was performed as described for Figure 5A.

Figure 5 Effects of NAMPT knockdown on chemotherapeutic agents

Mock or NAMPT knockdown PC3 cells were treated with paclitaxel (A) or etoposide (B) for two days at

concentrations indicated. Viable cells were counted as described for Figure 5A.

Figure 6 Effects of SIRT1 over-expression with NAMPT knockdown

(A) SIRT1 and NAMPT expression analysis in PC3 cells with different transduction combination. C+Scr:

empty expression vector(C) plus scrambled shRNA vector (Scr); C+shNAMPT: empty expression vector

plus shNAMPT vector; SIRT1+Scr: SIRT1 expression vector plus scrambled shRNA vector;

SIRT1+shNAMPT: SIRT1 expression vector plus shNAMPT vector. (B) Cell survival and proliferation

under treatment with hydrogen peroxide in mock (Top) or SIRT1 (Bottom) over-expressing cells with

scrambled and NAMPT shRNA knockdown. Viable cells were counted by trypan blue exclusion after

overnight H2O2 treatment. Limited protection by SIRT1 over-expression was seen with H2O2 lower than

25 μM.

Figure 7 Effects of SIRT1 and NAMPT double knockdown

Individual and combined knockdown of SIRT1 and NAMPT in PC3 cells were treated with PEITC and

H2O2 as described in Figure 5. Double knockdown did not further increase cell death.

Figure 8 Effects of SIRT1 knockdown on NAMPT over-expressing PC3 cells

Proliferation of PC3 cells co-transduced with empty expression vector(C) plus scrambled shRNA vector

(C-Scr), empty expression vector plus shSIRT1 (C-shSIRT1), NAMPT over-expression vector(NAMPT)

plus scrambled shRNA vector (NAMPT-Scr), or NAMPT over-expression vector plus shSIRT1

(NAMPT-shSIRT1). Cells were analyzed in the absence (A) or presence (B) of 100 μM hydrogen

peroxide.

Figure 9 Analysis of FOXO3a RNA and protein acetylation after FK866 treatment in PC3 cells.

(A) FOXO3a RNA analyzed by reverse transcription and real time PCR. Experiment was performed as

described for Figure 8A. (B) FOXO3a protein acetylation. Cells were harvested 24 h after FK866

treatment to avoid substantial FOXO3a degradation. FOXO3a was immunoprecipitated using FOXO3a

antibody. Western blot was probed with anti-acetyl lysine antibody. After stripping, the membrane was

re-probed with FOXO3a antibody.

Figure 10 Effects of AZD2281 on FK866 treatment

PC3 cells were treated with AZD2281 at concentrations 250-1000 nM alone or in combination with 20

nM FK866, with or without 200 μM H2O2 for 24 hours.

Normal liv

er

K562KCL-22

KCL-22

HepG2HCT-116

MCF-7

NNMT

GAPDH

A

B

NNMT

GAPDH

K562KCL-22

NPMNB MCF-7

MDA-MB-231

NC HCT-116

CaCO2

NL NC 209

H 460

Supplemental Figure 1

Supplemental Figure 2

Gel 1 Gel 2

Normal p

rostate tissue

Prostate cancer

Gel 3

250

130

72

52

28

GAPDH

NAMPT

GAPDH

KCL-22Norm

al PBL

K562

170

72

55

33

NAMPT

Normal

liver

HepG2

170

72

55

33

KCL-22

K562

KCL-22 ove

rload

BPH bearing tis

sue

Colon Colon Carcinoma

Lymph Node Lymphoma

Lung Lung Adenocarcinoma

Adrenal gland Liver

Pancreas Muscle

A

B

Grey matter Glioblastoma

Supplemental Figure 3

0500

100015002000

0 200H2O2 concentrations (uM)PC3 mock PC3 shNAMPT No.II

Viab

le C

ell n

umbe

r (x

103 )

Supplemental Figure 4

Viab

le C

ell n

umbe

r (x

103 )

0

500

1000

1500

0 20 40Paclitaxel concentration (uM)

PC3 Mock PC3 shNAMPT

02 0 04 0 06 0 08 0 0

1 0 0 01 2 0 0

0 5 1 0E to p o s id e ( u M )

M o c k s h P B E F

Viab

le C

ell n

umbe

r (x

103 )

shNAMPTMock

Supplemental Figure 5

0

50000

100000

150000

200000

0 12.5 25 50 100H2O2 (uM)

SIRT1-scr SIRT1-shNAMPT

0

50000

100000

150000

200000

0 12.5 25 50 100H2O2 (uM)

Mock-scr Mock-shNAMPT

B

A

C+ScrC+shNAMPT

SIRT1+Scr

SIRT1+shNAMPT

NAMPT

SIRT1

β-actin

Vectors

1 2 3 4

Mock over-expression

SIRT1 over-expression

Via

ble

cells

Via

ble

cells

Supplemental Figure 6

Surv

ival

rate

00.10.20.30.40.50.60.70.80.9

1

scramble shNAMPT shSIRT1 Double-KD

Untreated PEITC5 μM

PEITC10 μM

H2 O2100 μM

Supplemental Figure 7

0

0.5

1

1.5

2

2.5

3

0 1 2 3Day

C-Scr C-shSIRT1NAMPT-Scr NAMPT-shSIRT1

00.5

11.5

22.5

3

0 1 2 3Day

C-Scr C-shSIRT1NAMPT-Scr NAMPT-shSIRT1

PC3, No H2 O2

PC3, 100 μM H2 O2B

A

XTT

read

ing

XTT

read

ing

Supplemental Figure 8

Foxo3a RNA after FK866 treatment in PC3 cells

0

5

10

15

20

DMSO FK866

delta

Ct

DMSO

FK866

Supplemental Figure 9

Acetyl-Foxo3a

DMSO

FK-8

66

FOXO3a

Total lysate Input

FOXO3a IP

FOXO3a

A

B

0

100

200

300

400

500

600

700

800

900

1000

DMSO (-)

DMSO(H2O

2)

Fk866

(-)

Fk866

(H2O

2)

AZD-250(-)

AZD-250(H

2O2)

AZD-500(-)

AZD-500(H

2O2)

AZD-1000(-

)

AZD-1000(H

2O2)

FK+AZD25

0(-)

FK+AZD25

0(H2O

2)

FK+AZD50

0(-)

FK+AZD50

0(H2O

2)

FK+AZD10

00(-)

FK+AZD10

00(H

2O2)

viab

le c

ell c

ount

(x10

00)

Supplemental Figure 10