Scheme of PABA/NO effects on Ca²⁺/NO homeostasis in HL60 cells.

<p>PABA/NO effects start as extracellular NO-mediated (from spontaneous decomposition) surface protein-thiol modifications. Intracellular PABA/NO spontaneously (or under GSTP-catalysis) generates NO and stable nitro-aromatic compound(s) (PABA-SG). This can inhibit SERCA initiating a release of Ca²⁺ from the sarcoplasmic (endoplasmic) reticulum. The latter activates CaM and, consequently eNOS producing an NO burst. Increases of NO above threshold levels result in eNOS inhibition through its S-nitrosylation/glutathionylation. Once NO levels start to decline, spontaneous or catalyzed deglutathionylation of eNOS results in its reactivation to maintain steady state NO levels.</p

Inhibition of calmodulin diminishes NO generation following PABA/NO treatment.

<p>A) Basal NO levels in HL60 and TReNOS^−/− cells before and after incubation with 50 nM of the specific CaM-inhibitor W-7. B) Kinetics of NO generation in HL60, HL60 cells after preincubation (30 min) with 50 nM of W-7, and in TReNOS^−/− cells following PABA/NO (25 µM) addition. Data represent: mean ± SE (panel A) and representative traces (panel B) of 3 independent experiments.</p

The effect of eNOS suppression in HL60 cells after siRNA transfection.

<p>A) Fluorescent and DIC images of cells before (upper panels) and after (lower panels) transfection with FITC-labeled siRNA. B) Viability of mock (black column) and eNOS siRNA transfected cells (white column) detected by trypan blue exclusion. C) Immunoblot analysis of cell lysates (∼100 µg of total protein/lane) compared to standard (STD, Cayman) with eNOS detection. D) Immunoblot (panel C) quantification. E) Immunoblot analysis of eNOS in HL60 cells (1) and in HDMVEC (2) lysates (∼20 µg of total protein/lane); Data in B) and D) represent mean ± SE of 3 independent experiments. Loading controls with GAPDH detection on the same blot are presented.</p

PABA/NO effect on surface protein thiols and plasma trans-membrane potential in HL60 cells and controls for PABA, DETA/NO and DEA NONOate effects.

<p>A) Fluorescent detection (TG-1) of dose-dependent HL60 surface protein sulfhydryls level after PABA/NO, PABA and ThG addition. B). PABA (50 µM) addition to HL60 cells (with or without extracellular Ca²⁺) did not initiate generation of fluorescent metabolite (MW 622). C) Plasma trans-membrane potential (V_m) recorded as Bis-Oxanol emission at 520 nm after addition of 20 nM ThG, 25 µM PABA or 25 µM PABA/NO in HL60 cells. D) DETA/NO and DEA NONOate (25 µM) addition to HL60 cells did not initiate Ca²⁺ fluxes. Data represent: mean ± SE (panel A), representative traces averaged and smoothed with Sigma Plot 10.0 software (panel C), and original representative traces (panel B and D) of 3 independent experiments.</p

Estimated basal and PABA/NO-induced intracellular NO levels.

<p>Data represents mean±SEM for indicated number (N) of experiments.</p

PABA/NO treatment of HL60 cells results in temporal and dose-dependent increases in intracellular Ca²⁺.

<p>A) Time-dependent increases were assessed following treatment with 20 nM ThG; 25 µM PABA/NO; 25 µM PABA; 20 nM ThG after pretreatment with PABA/NO (15 µM, 30 min); 25 µM DEA NONOate B) Competitive inhibition of ThG (100 nM) - mediated increases in intracellular Ca²⁺ by PABA/NO, purified homogeneous nitro-aromatic product (MW 622 Da), or PABA. C) Dose-dependent increases in Ca²⁺ following PABA/NO (Control) or PABA were measured; in the presence of the intracellular Ca²⁺ chelator BAPTA-AM (5 µM); or in the presence of the extracellular Ca²⁺ chelator, EGTA (5 mM, long dashedline). The fluorescence measurements were recalculated as actual intracellular Ca²⁺ concentrations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014151#s4" target="_blank">Materials and Methods</a>). D) Kinetics of intracellular NO (left Y-axis) and Ca²⁺ (right Y-axis, solid line) generation after PABA/NO (15 µM) addition. Data are the average representative traces (A and D) or mean ± SE (B, C) for 3 independent experiments.</p

eNOS depletion alters basal and PABA/NO mediated NO generation and cell growth.

<p>A) HL60 and TReNOS^−/− cells labeled with DAF-FM to measure total basal NO levels. B). NO generation in HL60 cells following PABA/NO, PABA, or ThG titration. C) Kinetics of intracellular Ca²⁺ increase in TReNOS^−/− after PABA/NO (25 µM) or ThG (20 nM) addition. D) Growth curves for HL60 and TReNOS^−/− cells. Data represent: mean ± SE (panels A and B) for 3 independent experiments; representative traces of 3 independent experiments (panel C); and average ± variance for 2 independent experiments (panel D).</p

Reaction of PABA/NO with GSH <i>in vitro</i> and in HL60 cells.

<p>A) Scheme of nitro-aromatic PABA/NO-GSH adduct (MW 622 Da) generation. B) HPLC analysis of PABA/NO-GSH reaction <i>in vitro</i> and detection of PABA standard under the same conditions. C) HPLC analysis of HL60 lysate after incubation with 50 µM PABA/NO (1) and after “spike” with the purified nitro-aromatic adduct (MW 622 Da, 2). D) ESI MS analysis of purified from <i>in vitro</i> reaction nitro-aromatic PABA/NO-GSH adduct (m/z 623.8, left panel) and HL60 cells lysate after their incubation with purified nitro-aromatic adduct (m/z 623.8) product (right panel, indicated with asterisk).</p

PABA/NO-mediated S-glutathionylation of eNOS in HL60 and HDMVE cells.

<p>A) Control and PABA/NO-treated (25 µM, 30 min) HL60 cell lysates (∼100 µg protein/lane) resolved by non-reducing SDS-PAGE and evaluated by immunoblot with eNOS polyclonal primary antibodies and PSSG monoclonal primary antibodies and detected simultaneously with both: anti-mouse (red) and anti-rabbit (green) fluorescent secondary antibodies. B) Fluorescent detection (TG-1) of cytosolic protein sulfhydryls in HDMVE and in HL60 cells before (CTR) and after: PABA/NO (20 µM, 30 min) or ThG (200 nM, 30 min) addition. C) Immunoprecipitation of eNOS from HDMVEC with S-glutathionylated protein antibody. The blot was developed with the eNOS antibodies. Lanes are: 1- control HDMVEC lysate; 2- PABA/NO-treated (20 µM, 30 min) cell lysate; 3- IP of control cell lysate; 4- IP of PABA/NO-treated cell lysate; 5 -IP of PABA/NO-treated cell lysate after TCEP treatment. Arrows indicate low and high molecular weight eNOS bands. Loading controls are actin immunostaining. Data are: representative blots (panels A, C) and mean ± SD of 3 independent experiments, statistical significance p≤0.001.</p

PABA/NO-induced NO and fluorescent derivative generation kinetics in HL60 cells.

<p>A) PABA/NO (25 µM) addition to HL60 cells corresponds with an S-shaped generation of NO, becoming linear after cells are pretreated with 50 nM L-NAME for 30 min prior to PABA/NO (100 and 200 nM of L-NAME addition did not show any difference). DETA/NO and DEA NONOate (25 µM) addition to HL60 cells result in linear NO generation kinetics. B) Emission and excitation spectra of a purified fluorescent nitro-aromatic product (MW 622 Da) C) and D) Short and long exposure of HL60 cells to 25 µM PABA/NO produces linear kinetics for the fluorescent nitro-aromatic product (MW 622 Da) and S-shaped kinetics for NO generation. Data are: trace smoothed with Sigma Plot 10 software (panel A) and original traces (Panels B, C and D) representative of three independent experiments. Arrows indicate addition of drug.</p