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To determine whether superoxide and pCamKII were involved in at-level pain behaviors, an intrathecal catheter (CS-1 Intrathecal Catheter; ReCathCo, Allison Park, PA, USA) was implanted from the cisterna magna to the T5/6 level, and 2 cm of the free end was left exposed at the nape of the neck to allow drug administration. The catheter was implanted 5 days before the test under inhalation anesthesia (isoflurane; induction, 3%; maintenance, 1.5%). Saline (20 μL) was injected daily to prevent clogging of the intrathecal catheter. After implantation, the rats were individually housed and monitored for secure implantation. The superoxide anion scavenger Tempol (1 mg/10 μL/kg; Sigma-Aldrich, St. Louis, MO, USA), hydroxyl radical donor t-BOOH (0.4 mg/10 μL/kg; Sigma-Aldrich), non-specific ROS scavenger PBN (3 mg/kg; Sigma-Aldrich), pCamKII inhibitor KN-93 (50 μg/10 μL/kg), and inactive enantiomer KN-92 (50 μg/10 μL/kg; EMD Biosciences, San Diego, CA, USA) were intrathecally administered, and the i.t. tubing was flushed with saline (10 μL). To determine the contribution of AMPA and NMDA receptors to the hyperexcitability of WDR neurons, we administered the AMPA receptor antagonist NBQX (1 μg) or NMDA receptor antagonist MK-801 (50 μg) to SCI rats. All drug doses were set according to previous studies [10 (link),15 (link),24 (link)].

Three or more independent experiments were performed for all experiments. Animals and cell cultures for each group were randomized with the website www.randomization.com. Treatment, data collection, and data analyses were blinded by using different investigators or by masking sample labels.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.

Stress resistance assays were performed as described previously^{20 (link)}, with modifications. Gravid adults were allowed to lay eggs for 12 hrs on NGM plates seeded with OP50. For the oxidative stress assay, L4-stage worms were transferred onto 5 μM FUDR-treated NGM plates with E. coli bacteria and 7.5 mM tert-butyl hydroperoxide (t-BOOH, Sigma, St. Louis, MO, USA) solution. For the thermotolerance assay, L4-stage worms were placed in a 35 °C incubator. The number of live worms was counted every 2 or 3 hr and recorded as dead when the worms did not respond to tactile stimuli with a platinum wire. All the assays were conducted at least twice independently. OASIS (https://sbi.postech.ac.kr/oasis/) and OASIS2 (https://sbi.postech.ac.kr/oasis2/) were used for statistical analysis^{67 (link),68 (link)}, and p values were calculated using a log-rank (Mantel–Cox method) test.

To determine whether the effects of MEGR on nicotine sensitization were mediated by accumbal ROS, the ROS donor tert-butyl hydroperoxide (t-BOOH; 3.0 μg/200 nL/side; Sigma-Aldrich) [28 (link)] was dissolved in MRS and bilaterally administered into the NaccSh via the injectors (internal cannulae, 17 mm; 28-gauge) using motorized syringe pumps over a period of 60 s approximately 60 min after the fourth MEGR treatment. For the intra-NaccSh infusions of t-BOOH, stainless steel guide cannulae (15 mm; 23-gauge, 2 mm shorter than the internal cannulae) were bilaterally implanted into the brain using a stereotaxic instrument while the rats were under anesthesia; the cannula tips were placed 2 mm above the NaccSh so that the injectors exactly targeted the NaccSh. Five minutes after t-BOOH administration, the rats were systemically challenged with nicotine and tested in the locomotor testing boxes for 60 min. Immediately after the behavioral test, the rats were decapitated and their brains were removed to verify the guide cannula placement.

The manual oxidative stress assays were performed as described in detail in the bio-protocol⁶³ . L4 worms were manually picked onto fresh OP50 plates. The next day, 10–12 day-one old C. elegans were transferred into 24-well plates containing 1 mL M9 Buffer in quadruplicates for each strain and condition (three wells with sodium arsenite (Sigma-Aldrich) and one well M9 as control). For tBOOH stress assay, about 80 L4 C. elegans per condition were picked onto fresh RNAi plates. Three days later, 20 day-three-old C. elegans were picked onto NGM plates containing 15.4 mM tBOOH (Sigma-Aldrich). The survival was scored every hour until all animas died. Exploded animals were excluded from the statistics. The log-rank (Mantel–Cox) method was used to test the null hypothesis and calculate P values (JMP software v.9.0.2.).

K562 cells were seeded at 1.5 × 10⁵ cells/mL. A total of 24 h later, cells were treated with inhibitors and harvested after 24 h. Positive control cells were treated with 200 µM tert-butyl hydroperoxide (t-BOOH) (Sigma-Aldrich, Taufkirchen, Germany) for 30 min. Alkaline comet assay was performed as described in [19 (link)]. DNA damage (tail intensity) was evaluated with Comet IV software (Perceptive Imaging, Liverpool, U.K.). Per experiment, at least 50 cells were measured for each condition.