Supplementary Fig. 1

Supplementary Figure 1. Further JAG1 antibody characterisation.

(A) Binding specificity of hJAG1 mAbs for hJAG1 versus the hJAG2 ligand was also tested by

immunocytochemistry on cytospins of HEK293T transfectants. The expression of hJAG2 was

verified by a JAG2-specific mAb. (B) Key amino acids within the human JAG1 protein

(positions 199, 201, 203, 207 and 215) were mutated to alanine and soluble DSL-EGF3

recombinant proteins were used in dot blots to detect their binding to JAG1 mAbs. (C). Soluble

recombinant hJAG1 proteins were used to measure the dissociation constants (Kd) of JAG1

mAb binding: these comprised J1-DE3 (JAG1 DSL-EGF1-3); J1-NE3 (JAG1 N-terminus to

EGF3); J1-NE12 (JAG1 N-terminus to EGF12).

Supplementary Fig. 2

Supplementary Figure 2. Monoclonal antibody inhibition of JAG1 mediated Notch

signaling in tumor cell lines.

(A) FACS analysis showing expression of cell surface JAG1 (J1-183D mAb staining),

individual Notch receptors and JAG2 in a panel of human tumor cell lines. (B) qPCR analysis

showing the in vitro effect of JAG1-mAbs on Notch-target gene expression in the same panel

of cell lines described in (A). mIgG1 is the isotype-matched negative control for both mAbs,

DBZ is a pan-Notch inhibitor and DMSO is the corresponding negative control. Bar graphs

show the average ± SD of n = 5 (paired Student’s T-test; * P < 0.05, ** P < 0.01).

Supplementary Fig. 3

Supplementary Figure 3. Monoclonal antibody inhibition of JAG1 signaling in vascular

cells.

(A) FACS analysis showing J1-65D and J1-183D cell surface binding as well as cell surface

expression of individual Notch receptors and JAG2 on HUVEC. (B) qPCR analysis showing

the in vitro effect of J1-mAbs on Notch-target gene expression in HUVEC. (C) FACS analysis

showing J1-65D and J1-183D cell surface binding as well as cell surface expression of

individual Notch receptors and JAG2 on HUVSMC. (D) qPCR analysis showing the in vitro

effect of J1-mAbs on Notch-target gene expression in HUVSMC. For all in vitro treatments (B

and D), mIgG1 is the isotype-matched negative control for both mAbs, DBZ is a pan-Notch

inhibitor and DMSO is the corresponding negative control. N = 3 (paired Student’t T-test; * P

< 0.05, ** P < 0.01).

Supplementary Fig. 4

Supplementary Figure 4. Monoclonal antibody inhibition of JAG1 signaling in HUVEC-

HUVSMC co-culture.

(A) FACS analysis showing CD31 surface expression in HUVEC and HUVSMC. (B) Similar

analysis on co-cultured cells pre- and post-purification (1 representative experiment of 3 is

shown). (C) FACS analysis showing HUVSMC (CD31-) and HUVEC (CD31+) percentage after

48h co-culture (n = 3). (D) Co-cultured cell purity after CD31-based isolation (n = 3). (E) qPCR

analysis showing in vitro effect of J1-65D treatment on gene expression in co-cultured HUVEC

and HUVSMC. mIgG1 is the isotype-matched negative control for J1-65D. Graphs show the

average n = 3 (paired Student’s T-test; * P < 0.05, ** P < 0.01 and *** P < 0.001).

Supplementary Fig. 5

Supplementary Figure 5. Effect of anti-Notch pathway treatment on MDA-MB-231 2D

growth and J1-65D mAb titration and dose-dependent effect on 3D growth and gene

expression.

MTS assay showing the effect of various anti-Notch pathway treatments on MDA-MB-231

viability at low (A) and high (B) seeding density respectively. Bars show the average of 5

technical replicates from representative experiments. (C) Effect of different J1-65D mAb

concentrations on MDA-MB-231 spheroid growth. (D) qPCR analysis for the Notch-target

gene HES1 and the cytokine IL6 in treated spheroids in (A). One representative experiment

is shown in (C) and (D).

Supplementary Fig. 6

Supplementary Figure 6. J1-65D treatment of established OVCAR3 xenograft.

(A) J1-65D treatment has no effect on established OVCAR3 tumours (average ± SEM of n =

6/group). Individual tumor growth is shown in (B) for both groups. The small ticks on the x-

axis indicate days of treatment.

Supplementary Fig. 7

Supplementary Figure 7. J1-65D and J1-183D exhibit cross-reactivity with rat Jag1.

(A) Amino acid alignment showing differences between J1-65D and J1-183D mAbs putative

binding epitope in human, rat and mouse Jag1. (B) J1-65D and J1-183D surface labelling of

primary rat aortic smooth muscle cells (ASMC). (C) Effect of J1-65D and J1-183D mAbs on

Notch-target gene expression in primary rat ASMC in vitro. mIgG1 is the isotype-matched

negative control for both mAbs, DBZ is a pan-Notch inhibitor and DMSO is the corresponding

negative control. The average ± SD of n = 5 is shown (paired Student’t T-test; * P < 0.05, ** P

< 0.01, and *** P < 0.001).

Supplementary Fig. 8

Supplementary Figure 8. Anti-JAG1 treatment reduces MDA-MB-231-BR tumor growth

in a rat brain metastasis model.

(A) Jag1 expression analysis in MDA-MB-231-BR brain tumors shows positivity in tumor cells

(asterisk), blood vessels (arrow) and glial cells (arrowhead). (B) MRI-based analysis of the

animals presented in Figure 7 (6 per group) showing reduced tumor perfusion (CBF = cerebral

blood flow) at late time points in control animals, whilst normal contralateral brain showed no

difference (C) (average ± SD; 2way ANOVA). (D) Correlation analysis between volumes of T2

hyperintensity (surrogate of tumor size) and BBB breakdown or tumor normalized perfusion

(all MRI-based) shows significant correlations for control group, but none for hJ1-65Dv9-

treated animals (numbers in brackets indicate the week after tumor injection; correlation

analysis). * = P < 0.05; ** = P < 0.01; NS = no significance.