Tempol

CRISPR-mediated knockout plasmids containing guide RNAs targeting BAX, BAK1, NCKAP1, ACSL4, SLC7A11, CYFIP1, WAVE-2, Abi2, HSPC300 were generated in lentiCRISPR v2 (Addgene, #52961) according to the standard protocol. The SLC7A11 cDNA–containing expression construct was described in previous publications^{25 , 26} . The lentiviral construct expressing membrane-bound green fluorescent protein (mGFP) (#22479) and Rac1-Q61L cDNA-containing construct (#84605) were obtained from Addgene. NCKAP1 cDNA and shRNA constructs targeting RPN1, N-WASP, WHAMM were obtained from the Functional Genomics Core Facility of The University of Texas MD Anderson Cancer Center. NCKAP1 and Rac1-Q61L cDNA were subsequently cloned into the vector pLX302 with a C-terminal V5 tag (Addgene, #25896). WAVE-2 constructs were provided by Dr. Daniel D. Billadeau. All constructs were confirmed by DNA sequencing. The sequences of gRNAs and shRNA used in this study are listed in Supplementary Table 4. Necroptosis inhibitor Nec-1s (#2263) was from BioVision, and necrosis inhibitor Necrox-2 (#ALX-430-166-M001) was from Enzo. Ferroptosis inducer (1S,3R)-RSL3 (#19288) and apoptosis inducer staurosporine (#81590) were from Cayman Chemical. L-[1, 2, 1', 2'-14C]-cystine (#NEC854010UC) was from PerkinElmer. KL-11743 was from Kadmon. The following reagents were obtained from Sigma-Aldrich: 2-deoxy-D-glucose (#D8375-1G), Trolox (#238813), 4-Hydroxy-TEMPO (Tempol) (#176141), beta-mercaptoethanol (2ME) (#M6250), deferoxamine mesylate salt (DFO) (#D9533), ferrostatin-1 (#SML0583), chloroquine (#C6628), diamide (#D3648), diethyl-maleate (#D97703, BAY-876 (#SML1774), and L-Cystine (#C7602). All reagents were dissolved according to manufacturers’ instructions.

Animals were euthanatized with CO₂ and decapitated at 24 hr after sham operation or drug treatment for isolation and characterization of synaptic and nonsynaptic mitochondria, as described previously (Brown et al., 2006 (link); Naga et al., 2007 (link)). The spinal cords were rapidly removed and placed on an ice cold dissecting plate containing isolation buffer with 1 mM EGTA (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA), 20 mM HEPES, 1 mM EGTA; pH adjusted to 7.2 with KOH). The spinal cords were dissected into 2 cm segments centered on the injury site and homogenized in 2 mL of ice cold isolation buffer with EGTA. The homogenate was then centrifuged twice at 1,300g for 3 min at 4°C and the resulting supernatant removed and centrifuged at 13,000g for 10 min at 4°C. On the basis of pilot studies, mitochondria from two spinal cord segments were pooled in 500 μL of isolation buffer with EGTA to augment protein concentration for reliable mitochondrial respiration during experiments. The resulting crude mitochondrial/synaptosomal pellets were then placed atop a discontinuous Ficoll gradient (7.5%/10%) and centrifuged at 30,000g for 30 min at 4°C. The synaptosomal fractions were carefully removed from the interphase of Ficoll gradient and placed in a 2-mL centrifuge tube and the synaptic layer was washed with isolation buffer. The synaptosomal pellet was resuspended in 350 μL of isolation buffer with EGTA and burst in a nitrogen cell disruption chamber (1200 psi, 10 min) that was cooled to 4°C. The synaptosomal fractions were then applied for Ficoll purification again. The sedimented synaptosomal and nonsynaptosomal mitochondrial pellet was suspended in isolation buffer without EGTA and centrifuged for 10 min at 10,000g. The mitochondrial pellet was resuspended in EGTA-free isolation buffer at a concentration of ~10 mg/mL and stored on ice until further use. The protein concentration was determined with the BCA protein assay kit by measuring absorbance at 560 nm with a Biotek Synergy HT plate reader (Winooski, Vermont).

Hypertension was induced by angiotensin II (490 ng/kg/min) as described previously 25 (link) using either C57Bl/6 or mice transgenic for human SOD2 (tg^SOD2 mice). In addition, mice received a separate minipump for co-infusion of either TEMPOL, mitoTEMPO or vehicle as described in the figure legends. In other animals, mitoTEMPO treatment was started seven days after saline or angiotensin II minipump placement. Blood pressure was monitored using either the tail cuff method or telemetry as previously described 26 (link), 27 (link). Following 14 days of angiotensin II infusion the animals were sacrificed by CO₂ inhalation and aortas were extracted for the analysis of nitric oxide and O₂^∸ production, and endothelial functions. DOCA-salt induced hypertension was induced as described previously 28 (link) using C57Bl/6 mice. Ten days after surgery the mice were implanted with osmotic pumps containing saline or mitoTEMPO (0.7 mg/kg/day). Seventeen days after surgery the animals were sacrificed by CO₂ inhalation and segments of mouse aorta were used for analysis of vascular nitric oxide and O₂^∸ production. Endothelium-dependent vasodilatation was analyzed in isolated 3-mm aortic segments in organ chambers as we have previously described 27 (link).