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

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

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

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

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

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

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

Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis

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