Intracellular NCAMP-1 expression in zebrafish coelomic cells (ZFCC).

<p>ZFCC were fixed, permeabilzed and stained for intracellular NCAMP-1 (red) with rabbit anti-NCAMP-1 polyclonal antibody or normal rabbit IgG. Actin was stained with phalloidin (green) and nuclear morphology is indicated with DAPI staining (blue). (A) is DAPI only; (B) anti-NCAMP-1 only; (C) actin; and (D) are the merged images. E shows the negative isotype controls (DAPI + isotype control).</p

Western blot analysis of NCAMP-1 release from ATP treated zebrafish CC.

<p>ZFCC were untreated (NA) or treated for 30 min with 5 mM ATP (+A). Supernatants were harvested and analyzed by Western blot using rabbit polyclonal anti-NCAMP-1 antibody. (+) is the positive control of R-NCAMP-1-Histag binding. (-) is the negative control (e.g. no primary antibody).</p

NCAMP-1 or ATP do not cause cellular damage.

<p>(A) ZFCC were incubated with 1ug NCAMP-1 for 30min at room temperature and analyzed with YO-PRO-1 and PI. (B) 20mM and 30mM ATP were added in presence of YO-PRO-1 and PI and immediately analyzed (0min). Representative of 3 independent experiments.</p

Constitutive expression of NCAMP-1 in zebrafish tissues.

<p>Serial thin sections of zf trunk kidney and intestine were examined by H&E staining; rabbit polyclonal anti-NCAMP-1 serum and normal rabbit isotype control.</p

Binding of NCAMP-1 to ZFCC.

<p>Zebrafish coelomic cells were analyzed by flow cytometry in a two parameter histogram for their forward scatter and side scatter characteristics (PanelA). Panel B shows the ungated cells stained with 0.5 μg of Cy-3-NCAMP-1. The red histogram is the media (negative) control.</p

Soluble NCAMP-1 and ATP bind to ZFCC and induce a concentration dependent uptake of YO-PRO-1 dye.

<p>Indicated concentrations of soluble NCAMP-1 (A) or ATP (B) were individually added to a suspension of ZFCC at 5mM YO-PRO-1 and immediately analyzed by flow cytometry. Histograms are representative of three independent experiments per agonist at each concentration.</p

Cytotoxic activity of zebrafish effectors is increased following pretreatment of NCAMP-1 or ATP.

<p>HL-60 target cells were labeled with 5mM CFSE for 15 minutes at 37°C and washed. Targets were added to zebrafish effectors, pretreated for 30 minutes with either 5mM ATP, 1ug NCAMP-1, or no treatment, at an effector to target cell ratio of 8:1. Flow analysis was conducted at times 0, 1, 2, and 4 hours of co-incubation. Data points represent the mean ± standard error of 3 independent experiments. At times of 60 and 120 minutes, significant differences were observed between NCAMP-1 and media (** P≤ 0.01); NCAMP-1 and ATP *P≤ 0.05 as calculated by two-way ANOVA according to Bonferroni method.</p

NCAMP-1 and ATP bind to CC and induce an influx of Ca²⁺ ions.

<p>ZFCC were loaded with 4 uM Fluo-4 for 20 minutes at 37°C, washed with media, then analyzed for mean fluorescence intensity by flow cytometry. 50 seconds into analysis the calcium ionophore (positive control) or 1ug NCAMP-1 (A) or different concentrations of ATP (B) were added and analysis was continued. In C, cells were treated with the indicated ATP antagonist before addition of 30mM ATP and analyzed. Arrows indicate the addition of the agonist. Representative of 3 independent experiments.</p

YO-PRO-1 Uptake by ZFCC in response to NCAMP-1 treatment in the absence or presence of ATP.

<p>* P = ≤ 0.05 Different from NCAMP-1 only; Duncan’s (new) Multiple Range Test.</p><p>YO-PRO-1 Uptake by ZFCC in response to NCAMP-1 treatment in the absence or presence of ATP.</p

ATP induced YO-PRO-1 uptake by ZFCC is abrogated with P2X₇R inhibitors.

<p>30mM ATP was added to ZFCC with 5mM YO-PRO-1 and analyzed for percent fluorescence immediately or incubated with oxidized-ATP (oATP) (A), KN62 (B), or CBB (C) for 15 minutes prior to addition of ATP. Results are shown as the mean of 3 independent experiments ± S.E. * P≤ 0.01 significantly different from media controls as calculated by two-way ANOVA according to Bonferroni method.</p