O carboxymethyl hydroxylamine hemihydrochloride aoaa

In order to determine the effect of CBS, CSE and MPST in ccRCC tumors, RCC4 stable cell line (Sigma Aldrich, USA; passages 5–10) derived from clear cell renal cell carcinoma was used. Cells were grown in Dulbeco’s minimal essential medium (Sigma Aldrich, USA), supplemented with 10% fetal bovine serum and penicilin/streptomycin mixture. For experiments, cells were plated on 6-well plates or coverslips coated with polylysine and group of cells was treated with apoptosis inducer kit (AIK; Calbiochem, Merck, Darmstadt, Germany) diluted to 1:1000 as recommended by the producer. AIK is composed from the following inducers – Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, and Etoposide. Cells were treated also with D,L- propargyl glycine (PGG; 1 mM [21 (link), 22 (link)]; Cayman Chemicals), O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA; 10 μM [23 ]; Sigma Aldrich), or H₂S producing enzymes were silenced by the procedure described in Hudecova et al. [2 (link)] using SMARTpool ONTARGETplus CBS siRNA (Dharmacon, L-008617-00-0005), SMARTpool ON-TARGETplus Cth siRNA (Dharmacon, L-064123-01-0005), SMARTpool ONTARGETplus MPST siRNA (Dharmacon, L-010119-00-0005). As a negative control (scrambled), ON-TARGETplus NON-targeting Pool siRNAs were used (Dharmacon, USA).

Adult male CD1 mice with an average body weight of 23 g (20–25 g) were used in this study. Sodium hydrosulfide (NaHS), an H₂S donor, was obtained from Sigma-Aldrich (St. Louis, MO, United States) and dissolved in saline. O-(carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an H₂S inhibitor, was obtained from Sigma-Aldrich (St. Louis, MO) and dissolved in saline. For drug time effects assays, NaHS was intraperitoneally (i.p.) injected 30 min before or 30 min, 1, 2, 4, or 6 h after ICH. For drug dosage effects assays, NaHS (1, 10, 25, 50, and 100 μmol/kg) was i.p. injected 30 min before ICH. AOAA was i.p. injected 1 h before ICH. Animals were grouped into sham, ICH, ICH + NaHS, ICH + AOAA, and ICH + NaHS + AOAA groups. Sham-injured mice received craniotomy and injection with saline without ICH. All the animal procedures were approved by the Institutional Animal Use and Care Committee at Soochow University and conducted in accordance with the guidelines of animal use and care of the National Institutes of Health (NIH) and the ARRIVE (Animal Research: Reporting In Vivo Experiments). All efforts were made to minimize the numbers of animals used and ensure minimal suffering. In all experiments, data were obtained by investigators blinded to study group.

The CBS inhibitor O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) was obtained from Sigma-Aldrich (USA). Immediately after resolved in NS, AOAA was injected intrathecally at 10 μg/kg body weight, once daily for 7 consecutive days. Same volume of NS was used as control. L_5-6 DRGs from LDH rats after AOAA treatment were collected either for measurement of Na_V1.7 and Na_V1.8 expression or for patch clamping studies. For in vitro experiment, AOAA at 1 μM was incubated with acutely dissociated DRG neurons for one hour.