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Mohamedi et al. J Cancer Metastasis Treat 2019;5:37 I http://dx.doi.org/10.20517/2394-4722.2018.81 Page 5 of 15

antibodies from DAKO as the secondary antibodies. 3-3’-Diaminobenzidine was employed as a chromogen.
Selected slides were counterstained with hematoxylin to ascertain structural details. As immunostaining
controls, representative sections were processed in the same way as described above using nonimmune
rabbit or mouse sera instead of the primary antibodies or by omitting the primary antibodies during the
incubation. Furthermore, when available, additional controls were carried out using specifically preabsorbed
antisera.

Fibulin-1 immunostaining intensity was semiquantitatively evaluated by two independent observers directly
under the microscope using ten randomly selected fields (20x objective and a 20x ocular). The intensity of
the immunoreaction was evaluated as strong (+++), moderate (++), weak (+) or negative (-).

Survival analysis
To assess the effect of ADAMTS-1 and fibulin-1 on breast cancer prognosis, survival probability was
[36]
determined using the data available at www.kmplot.com . The results are presented as Kaplan-Meier long-
rank test survival plots.

Statistical analysis
Statistical analysis was carried out using Microsoft Office software. Data are presented as mean ± SE. The
occurrence of significant differences was determined with the Student-Welch t test. P values under 0.05 were
considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.005).

RESULTS
ADAMTS-1 and fibulin-1 interact in breast cancer cell lines
Fibulin-1 has been described as an ADAMTS-1 cofactor because of its proteolytic activity towards aggrecan
[19]
degradation . Fibulin-1 is not an ADAMTS-1 substrate; instead, it is an interacting partner that can affect
cancer-associated properties such as the migration, invasion and proliferation of breast cancer cell lines.
Thus, we used two common human breast cancer cell lines, MCF-7 and MDA-MB-231. First, we quantified
the endogenous expression of both proteins in cell extracts obtained from both cell lines [Figure 1]. No
detectable levels of fibulin-1 or ADAMTS-1 were visualized by western blotting in either the MCF-7 or the
MDA-MB-231 cell extracts [Figure 1A]. Thus, we transfected both cell lines with the corresponding cDNAs
(ADAMTS-1, fibulin-1 or both simultaneously) to obtain transfectants expressing one of the proteins or both
proteins together. For control purposes, we used an empty vector as a negative control [Figure 1A, control].
Once we obtained all the transfectants, we tried to probe the in vivo interaction of ADAMTS-1 and fibulin-1
in MCF-7 extracts by immunoprecipitation. To this end, we took advantage of the Flag epitope present in
the exogenously expressed ADAMTS-1 protein. Fibulin-1 western blotting following Flag (ADAMTS-1)
immunoprecipitation showed a 100 kDa reactive band only in extracts from the ADAMTS-1/fibulin-1
double-transfected cells [Figure 1B, top], which demonstrated the existence of this interaction in the MCF-
7 double transfectants [Figure 1B]. To probe the presence of ADAMTS-1 in the same immunoprecipitate, we
carried out a Flag western blot that showed the presence of a 100 kDa reactive band that corresponded to the
size of ADAMTS-1 [Figure 2, bottom].

Effect of the fibulin-1/ADAMTS-1 interaction on invasion properties
After we demonstrated the fibulin-1/ADAMTS-1 interaction in breast cancer cells, we decided to explore
the functional consequences of this event by first checking the effects of this interaction on the invasion
properties of both cell lines. To address invasion properties, we used Matrigel-coated invasion chambers
[Figure 2], and we observed that the fibulin-1/ADAMTS-1 interaction resulted in a significant reduction
in the invasion capability of both cell lines. Thus, the invasive potential of the double transfectants of both
the MDA-MB-231 and the MCF-7 cell lines was decreased compared with that of the control cell lines
(MDA-MB-231: average of 112 double transfectants cells/field vs. 240 control cells/field; MCF-7: 65 double