Supplemental Data

Notch Shapes the Innate Immunophenotype in Breast Cancer

Qiang Shen, Brenda Cohen, Weiyue Zheng, Ramtin Rahbar, Bernard Martin, Kiichi

Murakami, Sara Lamorte, Patrycja Thompson, Hal Berman, Juan Carlos Zúñiga-

Pflücker, Pamela S. Ohashi and Michael Reedijk

Supplementary Table 1. Notch-regulated cytokines† in HCC1143 breast cancer cells

siJ1 vs scr siN1/3 vs scr

Fold* Regulation Fold Regulation

CCL2 (MCP-1) 3.35 down 4.28 down

CXCL2 (MIP2A) 3.24 up 1.06 down

CXCL3 (MIP2B) 2.49 up 1.16 down

FGF2 (bFGF) 3.59 up 1.34 up

FGF19 5.92 up 1.34 down

IL1B 2.15 down 4.28 down

IL8 1.19 up 2.70 down

IL23A 1.94 down 2.61 down

IL28B 4.04 up 2.00 down

IL1F7 1.81 down 2.20 down

†Differentially-expressed cytokine genes following transfection of HCC1143 breast cancer cells with siRNA targeting JAG1 (siJ1), or combined NOTCH1 and NOTCH3 (siN1/3)

*Expression analysis using human whole genome (44K) Agilent microarray chips as described previously (1). Three replicated measurements were performed per group.

Supplementary Table 2. Primers and SequencessiRNA Company and catalogue # sequenceNotch 3 (pool of 3) Santa Cruz sc-37135 GUCAGAAUUGUGAAGUGAAtt

CUCGUCAGUUCUUAGAUCUttCCUCUCAUUUCCUUACACUtt

Jagged 1-55 Invitrogen HSS176255 CGCGACGAGUGUGACACAUACUUCANotch1-08 Invitrogen HSS107249 CCACCAGUUUGAAUGGUCAAUGCGAIL1β (pool of 3) Santa Cruz sc-39615 CCAGUGAAAUGAUGGCUUAtt

CCUGCGUGUUGAAAGAUGAttCACCUCUCCUACUCACUUAtt

CCL2 (pool of 3) Santa Cruz sc-43913 CCAGUCACCUGCUGUUAUAttCUCGCGAGCUAUAGAAGAAttGAAUCUGCAGCUAACUUAUtt

ASC (pool of 3) Santa Cruz sc-37281 CGAGGGUCACAAACGUUGAttCGGAAGCUCUUCAGUUUCAttCCCACCAAAUCAUCCUGAAtt

TGFβR1 (pool of 3) Santa Cruz sc-40222 GCAUGGUCAUGUUUGAAUAttCAAGAGUGCUGGACUUCUAttCUAGAACCUUUGAGUUACAtt

uPA (pool of 3) Santa Cruz sc-36779 CCACACACUGCUUCAUUGAttCCCAUGGUUGAGAAAUGAAttGUCUGAUUGUUAAGUCUAAtt

Capase-1 Santa Cruz sc-29235 GGGGCACAGGCAUGCCAAAshRNA SequenceHuman shJAGMouse shNotch3Mouse shJAG (3'utr)Mouse shJAG (1388)

CCGGCGTACAAGTAGTTCTGTATCTCGAGATACAGAACTACTTGCCGGGCCCTTTGAGTCTTCATACATCTCGAGATGTATGAAGACTCAAAGGGCTTTTTCCGGACATCTGCCAGCGGTCCTAATCTCGAGATTAGGACCGCTGGCAGATGTTTTTTGCCGGCGATGACTGTTCTCCAAATAACTCGAGTTATTTGGAGAACAGTCATCGTTTTTG

QRT-PCR Forward Primer Reverse PrimerHuman Actin cDNAHuman CCL2 cDNAHuman IL1β cDNAHuman IL10 cDNAHuman IL12 cDNAHuman JAG1 cDNAHuman Notch1 cDNAHuman Notch3 cDNAHuman TGFβ cDNAHuman TGFβR1 cDNAMouse Actin cDNAMouse Arg1 cDNAMouse CCL2 cDNAMouse CD206 cDNAMouse GAPDH cDNAMouse IL1β cDNAMouse IL6 cDNAMouse iNOS cDNAMouse JAG1 cDNAMouse Notch3 cDNAMouse TGFβ cDNAMouse TGFβR1 cDNA

CCACACTGTGCCCATCTACGACCATTGTGGCCAAGGAGATCTGTAACAGGCTGCTCTGGGATTCTCTTTCCTTGCTGGAGGACTTTAAGGGTAGTGGAGGCCTGTTTACCATTGGATGACCAGAATGGCAACAAAACCTGCCTGTCTGAGGTCAATCCTGCGATCAGGACATCAAACAATTCCTGGCGATACCTCAGCATCAGTGCACCCTTGTTACTTGGGAGGCTGTATTCCCCTCCATCGTGATTATCGGAGCGCCTTTCTAACTACAGCTTCTTTGGGACACTGCAGATGGGTGGGTTATTTGTGATGGGTGTGAACCACGGGTGTGTGACGTTCCCATTAATCCAGTTGCCTTCTTGGGACTGATCACCTTCGAGGGCAGCCGATGCTCACACCTGAAAGACCAACTGGACCTCGCTGTGAGACAGCCCGAAGCGGACTACTATCCTTGAGTCACTGGGTGTTATG

AGGATCTTCATGAGGTAGTCAGTCAGAGTTTGGGTTTGCTTGTCCAGGTGATTTCACTGGCGAGCTCAGGTACTTGTCTGGGTCTTGGTTCTCAGCTTAGGCCAGGCAACTCCCATTAGTTACTCATTACAGATGCCGTGGAGGGTCACAGTCGCACTTGTAGCAGGAGCAGGAAAAGGAGCGCTAAGGCGAAAGCCCTCAATTTAAGCCCTGCAGAGACTTCATACCAGTTGGTAACAATGCCATGTCCACACTGACTCTTCCATTCTTCATCCACGTGTTGGCTCAGGCATTGATGCTGCTGTTATGCAGTGAGCTTCCCGTTCACCTCCTGACCACTGTTGTTTCCTTGGATGGTCTTGGTCCTTAGCCATCCGTGGCAAAGCGAGCCAGGTGAATTTGCCTCCCGACTGCAGACACCACCATTGACACTTCCACATGTTGCTCCACACCCACTTAGCTGTCACCCTAATC

ChIP Forward Primer Reverse PrimerHA-1 TGTTTGTGGTCCCCTCTCTT TTGGATCCATCAGAAAAAGCHA-2 TGGAGAGCAAGTCCATGAAA GTGCTCGCTCTGCATTATCCHA-3 CTGTGTGTCTTCCACTTTGTCC TGACAATCGTTGTGCAGTTGuPA TTGCCTGCACAAATAAATGA CAGGGCTTGATAAGGATTGGCyclin D1 GAAACTTGCACAGGGGTTGT CTCAGCGACTGCATCTTCTTTGAPDH TACTAGCGGTTTTACGGGCG TCGAACAGGAGGAGCAGAGAGCGAGenotypingRBPJ F1 GTTCTTAACCTGTTGGTCGGAACCRBPJ R1 GCTTGAGGCTTGATGTTCTGTATTGCRBPJ R2 GGGCTGCTAAAGCGCATGCTRBPJ F3 CCTTGGTTTGTTGTTTGGGTTRBPJ R3 GTGGCTCTCAACTCCCAATCGTROSA5 GAGTTCTCTGCTGCCTCCTGRTTA3 AAGACCGCGAAGAGTTTGTCROSA3 CGAGGCGGATACAAGCAATARBPJ-HA F ATGACGGGGTCATTTACTCCRBPJ-HA R CAAGCGTAATCTGGAACATCMMTV-cre M046 CTGATCTGAGCTCTGAGTGMMTV-cre C031 CATCACTCGTTGCATCGACCPyMT1 GGAAGCAAGTACTTCACAAGGPyMT2 GGAAAGTCACTAGGAGCAGGG

E

D

A

B

C

Start codon

-1706

-2598

-2677

-2779

-3082-3160F

-3856G-3887H

-4688I

a t g t g c t g a g a c t c c c a c c c a g g a t g t t

t a g c t g t c t g c c t c c c a c t t c t g c t c t g

g t g a g t g g a a a t t c c c a c t c t g a g g a g g

a g c c a g g g a g c t c t c a c a t g g g g c t g t

t c g c t g c a g a c t c c c a c t c a g t g t c a gc a c t g c c t g g g c t c c c a c c a a c t t c t g c

t a c t c c t g a g c a t g g g a a t a g g g g t g g cg g c a t a g c a t a t g a g a a c a g a a a g a g t

t g c t g g c c a g g t g g g a a a a t a c t g a g c

Figure S1. Schematic Representation of the Human CCL2 Promoter/Enhancer Indicating the Location of Putative CBSBased on a search for the core binding motif TGRGAR (Nellesen et al., 1999), 9 low-affinity CBS (RTGRGAR; grey triangles, sites A - I) are identified. The 5’ locations of transcription factor binding sites are indicated relative to the start of translation of CCL2. The sequence of the minus strand is shown. Putative CBS are shown in light blue.

Supplementary Figure 1. Schematic Representation of the Human CCL2 Promoter/Enhancer Indicating the Location of Putative CBS Based on a search for the core binding motif TGRGAR (2), 9 low-affinity CBS (RTGRGAR; grey triangles, sites A - I) are identified. The 5’ locations of transcription factor binding sites are indicated relative to the start of translation of CCL2. The sequence of the minus strand is shown. Putative CBS are shown in light blue.

Caspase-1p50

p20

130-

175-NALP1

NALP390-

ASC22-

50-

22-

42- β-actin

b

c

Caspase-1p50

p20

50-

22-

ASC22-

42- β-actin

d

010203040506070

Sec

rete

d IL

1β(p

g/m

L)

* *

0

2

4

6

8

10

12

14

MDAMB231

THP-1

Nor

mal

ized

IL1B

mR

NA

exp

ress

ion LPS 0hr

LPS 3hrLPS 24hr

aA B

C D

Supplementary Figure 2. MDA MB231 Cells Contain Inflammasome Components Required For the Secretion IL1β A. IL1β mRNA expression in MDA MB231 and THP-1 cells was examined by qRT-PCR at 0, 3 or 24hr after stimulation with 100ng/ml of LPS. mRNA levels are expressed relative to untreated (0hr) MDA MB231 cells and are normalized according to the β-actin expression level. B. Immunoblot of inflammasome components NALP1, NALP3, ASC, Caspase-1 p50 and p20 after treatment of MDA MB231 cells with siscr, siIL1β, siN1/3 or siJ1. Mw markers are shown in kilodaltons. β-actin is included as a loading control. C. Immunoblot of ASC, Caspase-1 p50 and p20 after treatment of MDA MB231 cells with siscr, siASC or sicasp1. D. ELISA assay of secreted IL1β in MDA MB231 cells treated with siscr, siASC or sicasp1 (n=3/group). Bars represent standard error (SE). * P<0.05 compared with the control.

BAM1

M2

02468

10

M1 M2

IL1B

0369

1215

M1 M2

IL12

0

0.3

0.6

0.9

1.2

M1 M2

TGFβ

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

0

0.3

0.6

0.9

1.2

M1 M2

IL10

00.5

11.5

22.5

33.5 Arg1

0

40

80

120

160 CD206

0

2

4

6

8

10 IL1B

0.8

0.9

1

1.1

1.2 IL-6

0

15

30

45

60

75 iNOS

0

1

2

3

4 TGFβC

Figure S3. Generation of Human or Murine M1 and M2 Macrophages (A,B) (A) Morphology and (b) expression of cytokines characterizing M1 and M2 macrophages derived from THP-1 monocytic cells (Tjiu et al., 2009) exposed to PMA plus IL4 and IL13 (M2) or LPS and IFNγ (M1). (C) Bone marrow derived monocytes extracted from mouse femurs are transformed by exposure to M-CSF plus LPS and IFNγ to generate M1 macrophages (BM1) or IL-4 and IL-13 to obtain M2 macrophages (BM2), which express characteristic cytokines (Cho et al., 2014; Zhang et al., 2008).

Supplementary Figure 3. Generation of Human or Murine M1 and M2 Macrophages A. Morphology and B. expression of cytokines characterizing M1 and M2 macrophages derived from THP-1 monocytic cells (n=3/group) (3) exposed to PMA plus IL4 and IL13 (M2) or LPS and IFNγ (M1). C. Bone marrow derived monocytes extracted from mouse femurs are transformed by exposure to M-CSF plus LPS and IFNγ to generate M1 macrophages (BM1) or IL-4 and IL-13 to obtain M2 macrophages (BM2), which express characteristic cytokines(4,5).

M2 - -

β-actin42-

29-Pro-IL1β

-

MB231 sisc

r

siIL

1β

siC

CL2

sisc

r

siIL

1β

siC

CL2

sisc

r

sisc

r

sisc

r

sisc

r

sisc

r

siIL

1β

siC

CL2

sisc

r

siIL

1β

siC

CL2

- - -

1 2 3 4 5 6 7 8 9 10 11

Supplementary Figure 4. In Co-culture, Both M2 Macrophages and Breast Cancer Cells are a Source of Pro-IL1β Immunoblot of pro-IL1β from human MDA MB231 cells after treatment with siRNA targeting IL1β (si IL1β), CCL2 (siCCL2; negative control) or scrambled control (siscr), either in mono- or co-culture with THP-1-derived M2 macrophages. Mw markers are shown in kilodaltons. β-actin is included as a loading control.

M2

Tumor Cell

Latent TGF-β

LTBP

Large latent TGFβ complex

TGFβRII/I

ActiveTGFβ

uPA/uPAR

B

Notch

siscr siN1/3 siJ1siRNA

TGFβ 0Hr

3Hr

24H

r

0Hr

3Hr

24H

r

0Hr

3Hr

24H

r

uPA

p-Smad2/3

Smad2/3

A

β-actin

MB231 M2 -

uPA48-

42-

29-

29-

Pro-IL1β

CCL2

+ +

JAG1175-

+ ++

Anti-TGFβ - - +

0 0.5 1pixel density

MB231+M2

MB231+M2+Anti-TGFβ

C

Supplementary Figure 5. Notch Promotes TGFβ Signaling A. Immunoblot of uPA, phospho-SMAD2/3 and total SMAD2/3, after stimulation of MDA MB231 cells with TGFβ and treatment with the indicated siRNAs. B. Schematic demonstrating the production of large latent TGFβ (a complex between latent TGFβ and latent TGFβ binding protein (LTBP) by M2 cells and uPA/uPA receptor (R) plasmin/plasminogen complex-mediated conversion of latent TGFβ to its mature, active form (6). C. Immunoblot of JAG1, uPA, Pro-IL1β and CCL2 in MDA MB231 cells in mono-culture or in co-culture with THP-1-derived M2 macrophages, in the absence (-) or presence (+) of TGFβ neutralizing antibodies. Protein expression is quantified by pixel density, normalized to β-actin. Mw markers are shown in kilodaltons. β-actin is included as a loading control.

00.20.40.60.8

11.21.41.6

-Dox +Dox

Rel

ativ

e N

OTC

H3

mR

NA

0

0.20.4

0.6

0.8

11.2

1.4

-Dox +Dox

Rel

ativ

e JA

G1

mR

NA

***

00.05

0.10.15

0.20.25

0.30.35

0.4

-Dox +Dox

Tum

or w

eigh

t (g)

0

0.5

1

1.5

2

2.5

3

-Dox +Dox

Rel

ativ

e M

Ø in

filtra

tion

NS

NS

a

b

A

B

00.20.40.60.8

11.21.41.6

-Dox +Dox

Rel

ativ

e N

OTC

H3

mR

NA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-Dox +Dox

Rel

ativ

e JA

G1

mR

NA

***

00.05

0.10.15

0.20.25

0.30.35

0.4

-Dox +Dox

Tum

or w

eigh

t (g)

0

0.5

1

1.5

2

2.5

3

-Dox +Dox

Rel

ativ

e M

Ø in

filtra

tion

NS

NS

a

b

C

00.20.40.60.8

11.21.4

Rel

ativ

e M

Ø

infil

trato

n

*

-Dox +Dox +Dox w/IL1β+CCL2

Supplemenary Figure 6. Effects of Cytokine Rescue and Doxycycline Treatment on 4T1 TumorgraftsA. Relative mRNA levels of NOTCH3 and JAG1 in 4T1-luc shN/J1 tumorgrafts were examined by QRT-PCR and compared between groups without (-) or with (+) doxycycline (Dox) administration (n=6-8/group). Statistical significance is indicated by * P<0.05, ** P<0.005. B. Summary of flow cytometric analysis for macrophage (MØ; F4/80, CD11b) infiltration (corrected for per gram of tumor tissue) in 4T1-luc shN/J1 -Dox tumors, and +Dox tumors with or without recombinant mouse IL1β and CCL2 rescue. (n=5/group) C. Orthotopic 4T1 transplantations using parental 4T1-luc cells –Dox and +Dox. (n=3/group). At experimental endpoints, tumors were excised and compared for weight (left) and infiltrated macrophages (right). NS, non-significant.

0

0.5

1

1.5

2

-Dox +Dox

Rel

ativ

e N

eutro

phil

leve

ls

(CD

45+

CD

11b+

Ly6G

+ )

0

0.5

1

1.5

2

2.5

-Dox +Dox

Rel

ativ

e Tr

egle

vels

(C

D45

+C

D4+

foxp

3+ )

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-Dox +Dox

Rel

ativ

e C

D8

T ce

ll le

vels

(CD

45+

CD

8+C

D4- )

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-Dox +Dox

Rel

ativ

e C

D4

T ce

ll le

vels

(CD

45+

CD

4+C

D8- )

P=0.38

P=0.68

P=0.14P=0.08

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-Dox +Dox

Rel

ativ

e M

acro

phag

e le

vels

(C

D45

+C

D11

b+F4

/80+ )

*

Supplementary Figure 7. Analysis of Immune Infiltrates in 4T1-luc shN/J1 TumorgraftsSummary of flow cytometric analysis of macrophage, neutrophil, regulatory (Treg), CD8 and CD4 T cell infiltration (corrected for per gram of tumor tissue) in -Dox and +Dox 4T1-luc shN/J1 tumors (two experimental replicates; representative data from n=5/group shown).

Supplemental Methods

Datasets

Human breast cancer cell line expression data was obtained from the Genente(h

dataset (GSE12777) (7), and human breast cancer expression data was obtained from

the Sabatier et al. (GSE31448) (8).

Expression Array Analyses

Supervised clustering of normalized, median centered genes using an un-centered

correlation and average linkage matrix ordered by subtype of genes in the Notch-

activated and macrophage signatures in the two datasets, was conducted using Cluster

3.0. Heatmaps were generated using Java Treeview. Nearest mean centroids were

calculated by taking the mean of all signature genes in each tumor or cell line dataset.

The significance of relationships between gene expressions of the molecular subtypes

in the signatures, represented by the centroid were compared with the non-parametric

Dunn’s multiple Comparison post-test of the Kruskall-Wallis statistic using the Prism

software package. To calculate the correlations, JAG1, UPA, IL1β, CCL2, CASP1, ASC,

NALP1, NALP3 and NALP4 were individually compared to the nearest mean centroid of

the Notch-activated signature using the Spearman r correlation statistic in Prism. The

Notch activation and macrophage signatures in the breast cancer dataset were

correlated using the macrophage signature centroid as the gene phenotype label in a

gene set enrichment analysis (GSEA) of the Notch activation signature. Alternatively,

the macrophage signature centroid was correlated with the Notch activation signature

centroid using Spearman r correlation statistics.

Antibodies and Reagents

Notch1 (C-20), Jagged1 (H-114), Notch3 (M-134), IL1β (H-153), CCL2/MCP-1 (R-17),

TGFβR1 (V-22), Smad2/3 (C-8), p-Smad2/3, NALP1 (Nalpy1-4), HA (Y-11), CD163 (M-

96), β-actin, and horseradish peroxidase-conjugated secondary antibodies were

purchased from Santa Cruz Biotechnology. NALP3 (MAB7578) antibody, recombinant

human or mouse M-CSF, IL1β, CCL2/MCP-1, IL-4, IL-13, IFN-γ, and human or mouse

IL1β and CCL2/MCP-1 ELISA kits were from R&D Systems. Recombinant human

TGFβ1 was from PeproTech. ASC antibody (AL177) was purchased from AdipoGen.

Cleaved Notch1 (N1IC), TGFβ (3711S), Caspase-1 and RBPJ antibodies were from Cell

Signaling Technology. uPA antibody (MAB7776) was from EMD Millipore. GFP

antibody was purchased from Novus. Phorbol 12-myristate 13-acetate (PMA),

lipopolysaccharides (LPS), and gelatin were from Sigma-Aldrich. Human CCL2/MCP-1

antibody and active human IL1β receptor antagonist (IL1RA) were purchased from

Abcam. Lipofectamine RNAiMAX, calein AM, CellTrackerTM Red CMTPX, SYBR green

PCR master mix, penicillin-streptomycin, MatrigelTM basement membrane matrix (growth

factor reduced) and TGFβ-neutralizing antibody (1D11) were from Thermo Fisher

Scientific. Protease and phosphatase inhibitor cocktails were purchased from Roche.

The RNeasyPlus Mini Kit was from Qiagen. iScript cDNA synthesis and DC protein

assay kits were from Bio-Rad Laboratories. D-luciferin was from Cayman chemical.

Collagenase/Hyaluronidase Solution and DNase I were from Stemcell Technologies.

Adenoviruses, siRNAs and shRNAs

Adenoviruses expressing were constructed using the AdEasy system (Strategene).

cDNA containing full-length Notch1 intracellular domain (N1 IC) (kindly supplied by Dr. S.

Artavanis-Tsakonas), N3IC (kindly supplied by Dr. T. L. Wang), or LacZ control was

cloned into the pShuttle vector. Adenovirus vectors were then amplified and purified by

AD-293 packaging cells and Adeno-X purification kit (Clontech), respectively. siRNA

transfections were conducted in 6-well plates using 40nM siRNAs (Supplementary

Table S2) by Lipofectamine RNAiMAX according to the manufacturer’s protocol. For

inducible JAG1/Notch knock-down in breast cancer cell lines, specific shRNA

sequences for human or murine JAG1 and NOTCH3 (Supplementary Table S2) were

cloned into the lentiviral vector pLKO-Tet-On puromycin (Addgene) (9) or pLKO-Tet-On

vector modified to express eGFP. Lentiviral shJAG1eGFP or shNotch3puro particles

were produced using Addgene pMD2.G and psPAX2. Inducible cell lines (MB231/shJ1,

1143/shJ1, 4T1-luc shN/J1) were generated by infection followed by puromycin

selection or fluorescence activated cell sorting (FACS) of GFP positive cells.

Quantitative Real-Time (QRT) PCR

Primer sequences were designed using the IDT PrimerQuest Tool and are listed in

Supplementary Table S2. Total RNA was extracted using the RNeasyPlus Mini Kit

according to the manufacturer’s protocol. cDNA was prepared from 1μg of RNA using

the iScript cDNA synthesis kit and subjected to quantitative real-time PCR using the

default PCR cycle on a 7900HT Fast Real-Time PCR System (Applied Biosystems).

Amplified DNA products were detected and quantified by SYBR Green using Power

SYBR Green PCR Master Mix. Each sample was tested in triplicate for each primer set.

Dissociation curve analysis was also performed to ensure the absence of non-specific

amplification. After normalization according to β-actin expression level, depending on

individual experiments, QRT PCR values are expressed relative to MDA MB231 cells,

scr siRNA-treated cells, or tumor tissues with or without doxycycline, respectively.

Immunoblots

Cells or tumor tissue samples were homogenized in RIPA (25mM Tris pH 7.6, 150mM

NaCl, 5mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) lysis buffer freshly

supplemented with protease and phosphatase inhibitor cocktails. After the insoluble

components were pelleted at 15,000 x g for 3 minute, the concentration of proteins in

the supernatant was determined using the DC Protein Assay kit. Equal amounts of

proteins were then separated by 4-20% Tris-glycine gradient gels, unless otherwise

specified. Proteins in conditioned media were collected and concentrated as previously

described (1). For co-culture experiments, equal volumes of cell lysates were loaded on

gradient gels using β-actin as a loading control. Proteins were then transferred onto

PVDF membranes (Bio-Rad), and blotted with corresponding primary antibodies.

Following washing and incubation with HRP–conjugated secondary antibodies, the

proteins of interest were visualized in HyBlot CL films (Denville Scientific) using ECL

prime Western Blotting Detection Reagents (GE Healthcare). The films were then

scanned and quantified using ImageJ software for protein levels.

ELISA Assay

To determine IL1β and CCL2 expression levels in cell culture, following the indicated

treatments conditioned media were centrifuged at 500 x g for 10min and supernatants

collected to determine cytokine levels. IL1β and CCL2 in tumor tissues were released

by homogenizing the excised tissue samples, followed by a freeze-thraw cycle and

centrifugation for 10min at 4oC. Cytokine levels were analyzed using Quantikine ELISA

kits.

Mapping of CBS on the IL1β and CCL2 Promoters/enhancers

The FUZZNUC package in the EMBOSS software library (vers 6.0.0) (10) was used to

search a 5000-bp region upstream of the start codon of the human IL1B and CCL2

promoters/enhancers for a core motif TGRGAR (2,11). A position weight matrix (11)

was used to predict potential binding regions with the TFM-scan software package (vers

1.0) (12), with the 5’ locations of transcription factor binding sites are indicated relative

to the start of translation of IL1β and CCL2 (see Fig. 2A and Supplementary Fig. S1).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express kit

(Active Motif). Cells were fixed with 1% formaldehyde for 10 min, washed, lysed, and

sonicated to shear DNA to lengths ranging from 300 – 1200 bps. Chromatin/protein

complexes were immunoprecipitated with Rabbit anti-NOTCH1 antibody or Rabbit IgG

(#I5006, Sigma-Aldrich) for 18 hours at 4oC. Immunoprecipitates were reverse cross-

linked and treated with proteinase K. Precipitated chromatin samples were then

amplified with primer sets targeting potential CBS sites F, D and A and using primer

sets that target CBS sites in the Cyclin D1 and uPA promoters and the RNA

Polymerase II site in the GAPDH promoter as controls.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA) was performed with the Li-Cor Odyssey

Infrared EMSA kit. 5 μg of nuclear protein extract from MDA MB231 or MDA MB231

cells transfected with siscr or siCBF-1(RBPJκ) was incubated with 50 fmoles of double

stranded IRDye 700–labeled oligonucleotide probe. Where indicated, 10 pmoles of cold

wild type or mutant competitor oligonucleotide probe was added to the binding reaction.

Protein-DNA complexes were resolved by electrophoresis on 5% non-denaturing

polyacrylamide gels and detected using Li-Cor Odyssey Infrared Imaging System.

Monocyte Adhesion Assay

Human microvascular endothelial cells (HMVEC-L) were seeded onto gelatin-coated

24-well plates and grown to confluence. HMVEC-L monolayers were then pre-

incubated with cancer cell conditioned media (CM) for 16 hours, followed by the addition

of calcein-stained THP-1 monocytes (3 x 105 cells/well). After 1 hour, non-adherent

monocytes were removed, and adherent cells were quantified under fluorescent

microscopy.

Monocyte Extravasation Assay

HMVEC-L cells were seeded on basement membrane matrix-coated transwell inserts

(Corning Costar, 6.5mm diameter, 8μm pore size) and grown to confluent endothelial

monolayers. The luminal and subendothelial compartments were thus generated in the

upper and bottom chambers, respectively. HMVEC-L cells were then challenged with

conditioned media from MDA MB231 cells transfected with siRNAs as indicated. After

16 hours, the culture media in the upper chamber was removed and 200μL of RPMI

containing 4 x 104 calcein-stained THP-1 cells was added to the upper chamber for a

further 8 hours. Extravasation was stopped by removing the transwell inserts, and all

transmigrated monocytes were collected and counted under fluorescent microscopy.

Monocyte Differentiation and Co-culture Assays

Human M1- and M2-polarized macrophages were differentiated from THP-1 cells as

described by Tjiu et al. (3). Mouse M1- and M2-polarized macrophages were

differentiated from bone marrow-derived macrophages as previously described (4,5).

For co-culture experiments, basal-like human or mouse tumor cells were seeded with

M2 macrophages (ratio 3:1) into 6-well plates with serum-free RPMI media for 72 hours.

Mono-culture was performed in parallel by seeding the same number of cells as in co-

culture conditions. Experiments were repeated with tumor cells containing a stable

doxycycline-inducible shRNA-JAG1 in the presence or absence of 250ng/mL of

doxycycline.

Flow Cytometry

Tumors were minced and then digested overnight using digestion buffer (5% calf serum,

1 mg/ml of collagenase and 30 μg/ml of DNase I, 1% Penicillin-Streptomycin in

DMEM/F12). The digested samples were filtered through 70μm Falcon cell strainers

and the total viable cells determined. Murine immune cells stained with fixable viability

dye and fluorophore-conjugated antibodies targeting CD45, CD11b, F4/80, Ly6G, CD4,

CD8, FOXP3 (eBioscience) and CellTracker Red CMTPX-labeled cells were analyzed

with a BD Biosciences LSRFortessa Analyzer using FlowJo software (TreeStar).

Immunohistochemistry

Paraffin-embedded formalin fixed mouse mammary tumors sections were dewaxed, and

underwent heat active antigen retrieval. Sections were incubated with primary antibody

overnight at 4oC. Secondary antibody and ABC reagent (Vector labs) were added

sequentially and sections were developed with DAB reagent (Vector lab) and

counterstained with hematoxylin. To acquire WSI at 40× magnification, the stained

slides were scanned on a whole slide scanner (Nanozoomer 2.0-HT, Hamamatsu,

Japan). To quantifying macrophage infiltration, CD163 stained cells were counted from

random selected fields (480 x 430μm) in each tumor slide and the total cell number in

each selected field was calculated by ImageJ software. The percentage of infiltrated

macrophages was determined.

Statistics

Student’s t-test (two-tailed) was used for the statistical analysis of all experiments

(biological replicates), unless otherwise specified. Data are presented as mean ± s.e.m.

(error bars). P values < 0.05 were considered significance.

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