Figure Legends
Figure 1: A. alternata -induced Ca2+signaling in airway epithelial cells is blocked by C391. (A)Percentage of 16HBE14o- cells responding with
[Ca2+]i increases ≥ 150 nM within
the experimental time frame following A. alternata (± C391)
addition. (B - D) Representative population graphs from single
experiments depicting the average change in
[Ca2+]i in 16HBE14o- cells in
response to indicated compound plotted over time and shown with SEM in
the presence of: (B) 3 µM C391 alone; (C) A.
alternata EC50 alone and (D)EC50 of A. alternata combined with 3 µM C391.(E - H) Changes in
[Ca2+]i are shown as color maps
over time in response to A. alternata filtrate. Individual panels
are from (E) 20 sec prior to EC50 of A.
alternata filtrate addition and at (F - H) 60 sec intervals.
White lines approximate cell borders, color key for
[Ca2+]i is displayed in lower
right of first panel. (I - L) The same experiment as (E
- H ) excepting a 2 min preincubation with 3 µM C391 and continued 3 µM
C391 treatment during A. alternata filtrate exposure.(M) Percentage of 16HBE14o- cells responding with
[Ca2+]i increases ≥ 150 nM within
the experimental time frame in response to the P2Y agonist ATP (2.5 µM)
or the PAR1 agonist Thrombin (100 µM) ± C391. (N-Q)Representative population graphs from single experiments depicting the
average change in [Ca2+]i in
16HBE14o- cells in response to indicated compound plotted over time and
shown with SEM in the presence of: (N) 2.5 µM ATP alone;(O) 2.5 µM ATP and 3 µM C391 (P) 100 µM Thrombin;(Q) 100 µM Thrombin and 3 µM C391.
Figure 2: A. alternata -induced MAPK signaling in airway
epithelial cells is inhibited by C391. (A) MAPK
phosphorylation (p-MAPK) undergoes a concentration-dependent increase in
response to the potent and selective PAR2 agonist
2at-LIGRL-NH2. (B) MAPK phosphorylation
(p-MAPK) undergoes a concentration-dependent increase in response toA. alternata filtrates. (C) Concentration response curve
for C391 block of A. alternata (20 µg/mL)-induced MAPK
phosphorylation; IC50 is noted in the text. For all
experiments, 3 ≤ n ≤ 4. * indicates difference from HBSS-treated control(A, B) or from A. alternata -treated control(C) , p ≤ 0.05.
Figure 3: Activation-dependent β-arrestin-2 recruitment to PAR2
is inhibited by C391. Bioluminescence resonance energy transfer (BRET)
was measured upon addition of: (A) the potent PAR2 agonist
2-furoyl-LIGRLO-NH2 (1 µM) or (B) A.
alternata filtrate (Alt ; 65 µg/mL) and increasing concentrations
of C391. C391 effectively blocked β-arrestin-2 recruitment following
PAR2 activation via peptidomimetic or A. alternata proteinase. n
≥ 3 for all experimental time points; IC50s are noted in
the text.
Figure 4: C391 reduces asthma indicators in a C57Bl/6 murine
model. (A - F) Typical H&E staining of C57Bl/6 lungs treated
with: (A) HBSS; (B, E) A. alternata ;(C) A. alternata + 0.25 nmoles C391 or (D, F)A. alternata + 2.5 nmoles C391. Images A-D were imaged with 10X
and E-F were imaged with a 40X objective. A. alternata induces
significant inflammation (increased cellular staining) that is reduced
by low concentrations of C391 and mostly eliminated by higher
concentrations of C391. (G - J) Typical Alcian Blue staining of
C57Bl/6 lungs treated with: (G) HBSS vehicle control;(H) A. alternata ; (I) A. alternata +
0.25 nmoles C391 or (J) A. alternata + 2.5 nmoles C391.(K - L) Semi-quantitative histological grading of lung
inflammation and Alcian Blue staining shows a significant increase in
cellular infiltrate (K ), thickening of the basement membrane
(L ) % bronchial epithelial cells displaying positive mucus
staining (M ), that are significantly reduced by either low
(0.25 nmoles) or high (2.5 nmoles) C391 concentrations. n = 3 for all
experiments, * indicates significant difference from HBSS control, #
indicates significant difference from A. alternata -induced
changes; p ≤ 0.05.
Figure 5: C391 reduces asthma indicators in BALB/c murine
model. (A - D) Typical H&E staining of BALB/c lung slices
from animals treated with: (A) HBSS vehicle control;(B) A. alternata ; or (C) A. alternata +
5 nmoles C391. (D) Semi-quantification of H&E staining.
Typical PAS staining of BALB/c lung slices from animals treated with:(E) HBSS vehicle control; (F) A. alternata ;(G) or A. alternata + 5 nmoles C391. (H)Semi-quantification of PAS staining. A. alternata treatment
induces significant increases in inflammation and mucus production that
is reduced by C391. B denotes bronchioles, M denotes mucus plug, simple
arrows indicate inflammatory cell staining and closed arrows are
directed at goblet cell staining. n ≥ 4 for all experiments; * indicates
significant difference from HBSS control; # indicates significant
difference from A. alternata -induced staining; p ≤ 0.05.