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.