Supplementary Materials and Methods
Flow cytometry - Single-cell suspensions were generated from mouse bone-marrow
samples and stained with the following antibodies; from eBioscience anti-CD4 (RM4-5),
anti-TCRβ (H57-597), anti-CD45.1 (A20), and anti-CD45.2 (104). All samples were
analyzed using a Fortessa flow cytometer (BD Biosciences) and data were analyzed using
FlowJo software (Treestar).
G-protein assay - To determine the G-protein involved in the matriptase dependent
activation of PAR-2 and NFkB, inhibitors of Gq/11, G12/13 and Gi were utilized,
taking advantage of fusion proteins including regulator of G protein signaling-homology
(RGS) domains targeting these G protein  subunits. For the Gq/11 assay the
mammalian expression vector pCEFL-EGFP containing cDNA encoding dominant
negative RGS-βARK mutant targeting Gq/11 was used. For the G12/13 assay the
expression vector pCEFL-myc-mCherry containing dominant negative RGS-rhoGEF
mutant targeting G12/13 was used http://www.ncbi.nlm.nih.gov/pubmed/21934106. In
both cases the cells were also transfected with either SRE-luciferase or NFkB-luciferase
reporter together with pRL-Renilla and PAR-2 as described above. The respective empty
vector was used as control. The transfection medium was changed after 12 h, and 36 h
after transfection and the cells were serum-starved overnight.
Test of dominant negative constructs – To verify that the dominant negative RGS-βARK
mutant targeting Gq/11 and the dominant negative RGS-rhoGEF mutant targeting
G12/13 were functional, inducible activators of the respective pathways were utilized.
Cells were transfected G protein coupled receptors activated solely by synthetic ligands
(RASSL),
GαqRASSL
to
activate
the
Gαq/11
http://www.ncbi.nlm.nih.gov/pubmed/23177739 pathway or GαiRASSL and Gα13i5 to
activate the Gα12/13 pathway http://www.ncbi.nlm.nih.gov/pubmed/21934106, in
combination with SRE-luciferase reporter, pRL-Renilla and either dominant negative
mutant or empty vector. 36 h post-transfection the cells were serum starved ON. The next
day the cells were treated with a final concentration of 1 µM CNO to activate the GRASSL in serum-free media for 6h before the luciferase activity was determined.
HGF activation and HGF Western blot assays
HaCat cells were plated in 6-well plates and grown in DMEM supplemented with 10%
FBS for 24 h. Following 12 h of serum starvation, cells were treated with 100 ng/ml
hepatocyte growth factor (R&D Systems) or 10 µM PAR-2 agonist peptide ([3H]2furoyl-LIGRL-NH2) (Tocris) for 15 min. HEK293 cells were plated in poly-L-lysinecoated 6-well plates and grown in DMEM supplemented with 10% FBS for 24 h. Cells
were transfected with SRE-luciferase reporter together with pRL-Renilla and either PAR2 or the empty vector. 24 h post transfection, cells were starved ON and treated with
either HGF for 5 min and 15 min or recombinant matriptase for up to 3 h. Western
blotting was performed as described previously (Szabo et al., 2012). Briefly, after the
cells were lysed, the protein concentration was determined by the BCA assay (Pierce,
Rockford, IL) and 40 μg of total protein was loaded onto 4–12% reducing sodium
dodecyl sulfate–PAGE gels and analyzed using a rabbit monoclonal anti-human phosphoc-Met, rabbit monoclonal anti-human c-Met, rabbit monoclonal anti-human phosphoGab1, rabbit monoclonal anti-human Gab-1 (all purchased from Cell Signaling
Technology). Anti-human -tubulin (Santa Cruz, Dallas, TX) primary antibody was used
as a loading control.
Supplementary Figure 1. Generation of bone marrow chimeras.
At the same time as experimental bone marrow chimeras were generated, additional
chimeric mice were generated to validate the procedure. Non-transgenic control mice
were lethally irradiated and congenic CD45.1 bone marrow was transferred. Mice were
euthanized after eight weeks and the frequency of host CD45.2+CD45.1+ and donor
CD45.1+ bone marrow was examined by FACS. (A) Plot shows CD45.2 versus CD45.1
staining on bone marrow cells isolated from chimeric mice. (B) Plot shows TCR versus
CD4 staining gated on host bone marrow cells (top) or donor bone marrow cells (bottom)
and show frequencies of fully differentiated T cells within the bone marrow population.
Data
are
representative
of
three
individual
chimeric
mice.
Supplementary Figure 2. Gq11 and G12/13 are dispensable for matriptase-PAR2-mediated NFκB activation.
HEK293T cells were transfected with empty vector (bars 1 and 2) or PAR-2 expression
vector (bars 3-8), serum response element-luciferase (A and C) or NFκB-luciferase (B
and D) reporter plasmids. Cells in A and B were transfected with pCEFL-GFP containing
dominant negative RGS-ARK mutant targeting Gq/11. Cells in C and D were
transfected with pCEFL-myc-mCherry containing dominant negative RGS-rhoGEF
mutant targeting G12/13. The transfected cells then were treated with either vehicle
(bars 3-4), 10 M PAR-2 agonist (PAR-2 ag., bars1, 2, 5 and 6) or 15 nM recombinant
human matriptase (rhMat) (bars 7 and 8) for 6 h. All cells were transfected with a pRLRenilla luciferase reporter plasmid as an internal control for transfection efficiency. Data
are shown as the mean ± standard deviation of the mean of triplicate transfections of
firefly luciferase light units/Renilla luciferase light units. The data are representative of
three similar experiments. E and F. Fluorescence microscopy demonstrating GFP
expression in cells transfected with dominant negative pCEFL-RGS-ARK or the
respective empty vector (E) or demonstrating mCherry expression in cells transfected
with pCEFL-myc-mCherry-RGS-rhoGEF or the respective empty vector. G and H.
HEK293 cells were transfected with GqRASSL and RGS-βARK or empty vector (G) or
GiRASSL and G13i5 and RGS-rhoGEF or empty vector (H). Data are shown as the mean
± standard deviation of the mean of triplicate transfections of firefly luciferase light
units/Renilla luciferase light units.
Supplementary Figure 3. PAR-2 and pro-HGF activation by dysregulated
matriptase elicit different pro-tumorigenic signaling pathways.
(A) Enumeration of mast cell accumulation in 44-week-old c-Met-sufficient K5-Mat+/0
(mix of K5-Mat+/0;K14-Cre;Hgfr+/+,+/-,+/fl and K5-Mat+/0;Hgfr+/+,+/-,+/fl,-/fl, N=7, red circles),
c-Met-deficient K5-Mat+/0 (K5-Mat+/0;K14-Cre;Hgfr-/fl, N=8, green circles), and control
(mix of K14-Cre;Hgfr+/+,+/-,+/fl, N=7, blue circles) mice. * P < 0.01, ** P < 0.002, N.S. =
not significant (Mann-Whitney U-test, two-tailed). (B-D) Representative examples of
toluidine blue-stained dorsal skin sections from 44-week-old littermate K5-Mat+/0;K14Cre+/0;Hgfr+/- (left panel), K5-Mat+/0;K14-Cre+/0; Hgfrf/- (second panel from left), and
K14-Cre+/0;Hgfr+ (right panel) mice. Examples of dermal mast cells (blue) are indicated
with arrowheads. Size bars = 50 m. (E) HaCat cells were treated with vehicle (lane 1),
the PAR-2 agonist, 2-furoyl-LIGRO-amide (10 M) (lane 2) or recombinant HGF (2.5
n) (lane 3) for 15 minutes. Cell lysates were subjected to SDS/PAGE and Western
blotting using antibodies against total c-Met (top panel), phosphorylated c-Met (second
panel from top), total Gab-1 (middle panel), phosphorylated Gab-1 (second panel from
bottom), and tubulin (bottom panel). Positions of molecular weight markers (kDa) are
indicated on the left. (F) HEK293 cells were mock-transfected (lanes 1, 3, 5, and 7) or
transfected with a PAR-2 expression vector (lanes 2, 4, 6, and 8). Thereafter, the cells
were treated with vehicle (lanes 1 and 2), with 2.5 nM HGF for either 5 min (lanes 3 and
4) or 15 min (lanes 5 and 6) or with 15 nM soluble recombinant matriptase for 3 h (lanes
7 and 8). Cell lysates were analyzed by Western blotting using antibodies against total cMet (top panel), phosphorylated c-Met (second panel from top), total Gab-1 (middle
panel), phosphorylated Gab-1 (second panel from bottom), and tubulin (bottom panel).
Positions of molecular weight markers (kDa) are indicated on the left.