Corm 2

Calcium-free phosphate buffered saline (PBS), CORM-2, CORM-3, ruthenium chloride and carboplatin were obtained from Millipore Sigma (Saint Louis, MO, USA). Venoms dissolved in PBS (50 mg/mL) were obtained from archived, never thawed aliquots maintained at −80 °C in the laboratory that were used in previous investigations [1 (link),2 (link),3 (link),4 (link),11 (link),36 (link)]. Bothrops moojeni and Calloselasma rhodostoma venoms were obtained originally from the National Natural Toxins Research Center at Texas A&M University (Kingsville, TX, USA). Additionally, Echis leucogaster, Heloderma suspectum, Oxyuranus microlepidotus and Pseudonaja textilis venoms were originally purchased from Mtoxins (Oshkosh, WI, USA). Calcium chloride (200 mM) was obtained from Haemonetics Inc., Braintree, MA, USA. Pooled normal human plasma (George King Bio-Medical, Overland Park, KS, USA) that was sodium citrate anticoagulated and maintained at −80 °C was used.

Tunicamycin (Tm), MitoTEMPO, and CORM-2 were from MilliporeSigma (Billerica, MA, USA).

CORM-2, DMSO, RIPA and LPS were purchased from Sigma-Aldrich (MO, USA). CORM-2 was solubilized in DMSO to obtain a 40 mmol/L stock. An inactive form of CORM-2 (negative controls) was used in some experiments and prepared as follows: iCORM-2 was inactivated form of CORM-2 by leaving the stock of CORM-2 at 37°C in a 5% CO₂ humidified atmosphere for 24 h to liberate CO. The iCORM-2 solution was bubbled with nitrogen to remove the residual CO present in the solution. 1 × or 10 × HBSS with or without Ca²⁺ Mg²⁺, RMPI 1640 medium, fetal bovine serum (FBS) and agarose were purchased from Life Technologies (CA, USA). Antibodies to p38 mitogen-activated protein kinase (MAPK) and p-p38 MAPK and the p38 MAPK inhibitor SB203580 were purchased from Cell Signaling Technology (MA, USA). Antibodies to FPR1 and G protein-coupled receptor kinase 2 (GRK2) and the GRK inhibitor 4-amino-5-(bromomethyl)-2-methylpyrimidine hydrobromide were purchased from Santa Cruz Biotechnology (TX, USA). All other chemicals were of reagent grade and obtained from Sigma unless otherwise stated.

CORM-2 from Sigma (St Louis, MO. USA) was dissolved in dimethyl sulfoxide (DMSO) to obtain a 40 mM stock with inactive form (iCORM-2) of the compound prepared as described previously [16 (link)] and used as a negative control in some experiments. Anti-zonula occludens protein-1 (ZO-1), claudin-1 and occludin from Santz Cruz Biotechnology (Santa Cruz, CA); anti-total-myosin light chain (MLC) and anti-phospho-MLC (p-MLC) from Cell Signaling Technology (Danvers, MA, USA); anti-β-actin from KeyGen BioTECH (Nanjing, China) were used in this study. Enzyme-linked immunosorbent assay (ELISA) kits from Hermes Criterion Biotechnology (HCB, Vancouver, Canada) was used.

Tricarbonyldichlororuthenium (II) dimer (CO-releasing molecule-2, CORM-2) purchased from Sigma-Aldrich Canada (Cat #: 288144, Oakville, Ontario, Canada) was dissolved in DMSO to make a 50 mM stock solution and kept at −20°C for short-term storage or −80°C for long-term storage respectively. Inactive CORM-2 (iCORM-2) was prepared by dissolving CORM-2 in DMSO to make 1 mM solution, and kept for 3 days at room temperature. It was diluted with equal amount of DMEM and kept at 37°C for 24 h before use.