S nitrosoglutathione

Ribostamycin, PDI, BfA, GM1, GSH, CTA, S-nitrosoglutathione, and anti-CTB antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Bacitracin was purchased from Calbiochem (La Jolla, CA), CT was from List Biological Laboratories (Campbell, CA), and phosphate-buffered saline (pH 7.4) with 0.05% Tween 20 (PBST) was from Medicago (Uppsala, Sweden). ERp57, the anti-ERp57 antibody, and the anti-ERp72 antibody were from Abcam (Cambridge, MA). ERp72 and the anti-PDI antibody were purchased from Enzo Life Sciences (Farmingdale, NY). The anti-CTA1 monoclonal antibody 35C2 [52] (link) was a generous gift from Dr. Randall K. Holmes (University of Colorado School of Medicine). The pOLR130 plasmid encoding mature human PDI with an N-terminal His₆ tag [53] (link) was generously provided by Dr. Lloyd Ruddock (University of Oulu, Finland). Uniformly ¹³C-labeled ¹³C₆-_D-glucose and D₂O were purchased from Cambridge Isotope Laboratories (Andover, MA). Uniformly ¹³C-labeled CTA1-His₆ was produced as described in [7] (link) and purified as described in [22] (link).

S-nitrosoglutathione and H₂O₂ were purchased from Sigma-Aldrich, St. Louis, MO, USA.

E. histolytica trophozoites (1×10⁶) in TYI-S-33 medium (without serum) were exposed to 350μM S-nitrosoglutathione (Sigma-Aldrich, St. Louis, MO, USA) for two hours at 37°C. The viability of the trophozoites was determined by the eosin dye exclusion method[41 (link)].

Acetaminophen (APAP; 4-acetamidophenol), Percoll, Hepes, (heparin sodium salt grade I-A from porcine intestinal mucosa), penicillin G (sodium salt), RPMI-1640 modified media (with L-glutamine and without sodium bicarbonate and phenol red), 0.4% trypan blue solution, GSH, GSSG, S-nitrosoglutathione (GSNO), 3NT, NADH, NAD, and trifluoperazine (TFP; 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine), ATP Bioluminescent Assay kit, were products of Sigma Chemical Company (St Louis, MO). Collagenase A from Clostridium histolyticum was purchased from Roche Diagnostics (Indianapolis, IN). MitoSOX Red, 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), and JC1 were obtained from Life technologies (Eugene, OR). LDH (lactate dehydrogenase) cytotoxicity detection kit was a product of from Roche Diagnostic Corporation (Indianapolis).

NO donors with different half-lives in pH 7.4 buffer tested in this study are listed in Table 1. Sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO), MAHMA NONOate (NOC-9), PROLI NONOate and Spermine NONOate (S150), diethylamine NONOate sodium salt hydrate (DEA NONOate) and carboxy-PTIO potassium salt (PTIO) were purchased from Sigma Aldrich, UK. SNP and RSNOs stock solutions were prepared in phosphate saline buffer (pH 7.4), with SNP prepared and kept in dark. NONOates stock solutions were prepared in 0.01 M NaOH. All stock solutions were filter sterilized and diluted into fresh M9 media on ice. During preparation procedures, all solutions were kept on ice before use and SNP was kept in the dark. All treatments were conducted at 37°C, with SNP exposed to light.Table 1

NO donors tested in this study and their half-lives at 37°C, pH ~ 7.4

SNP	< 2 mins
SNAP	6 h
GSNO	1–3 h
PROLI NONOate	1.8 s
MAHMA NONOate	1 min
Spermine NONOate (S150)	39 min
DEA-NONOate	2 min

S-nitrosoglutathione (GSNO), metronidazole, H₂O₂, and G418 were purchased from Sigma-Aldrich, St. Louis, MO, USA. A stock solution of S-nitroso-L-cysteine (CysNO) was prepared according to a previously reported method64 (link) in which 0.2 M NaNO₂ is mixed with 0.2 M cysteine in HCl, and the mixture is then neutralized using NaOH [30]. The concentration of CysNO was determined spectrophotometrically using an extinction coefficient of 900 M⁻¹cm⁻¹. Under these conditions, the yield of CysNO was more than 90%.