Fig 2: OPA effects upon mitochondrial oxygen consumption, ATP production, and mitochondrial membrane permeability.(a, b) Agilent Seahorse cell mitochondrial stress test results measuring oxygen consumption rate (OCR) in NCI-H1703 parental cells (a) and shControl versus shHIGD2A/COX5A dual knockdown (b) NCI-H1703 cells. Cells were pre-treated with either DMSO vehicle or OPA for 30 min prior measuring basal OCR for approximately 20 min prior to H+-ATPase inhibitor oligomycin (2.5 μM) treatment. ATP-linked respiration and proton leak were measured for approximately 20 min prior to mitochondrial uncoupler FCCP (2 μM) treatment. Maximal respiratory capacity recorded for 20 min prior to the experiment concluding with complex I inhibitor Rotenone and complex III inhibitor Antimycin A (0.5 μM) treatment. Data shown are average ± sem, n=7 biologically independent samples/group. (c) ATP production rate time-course study in NCI-H1703 cells treated with DMSO vehicle or OPA. Cells were treated with either DMSO vehicle or OPA 2.5 h prior to oligomycin (1.5 μM) treatment. Kinetic study concluded when Rotenone and Antimycin A (0.5 μM) were added. Data shown are average ± sem, n=7 biologically independent samples/group. (d) NCI-H1703 cells were treated for 2 h with OPA and other positive control mitochondrial inhibitors Oligomycin (2.5 μM), FCCP (2 μM), Rotenone/AA (0.5 μM), and CCP (10 μM), before being labeled with tetramethylrhodamine methyl ester (TMRM), a dye that accumulates in mitochondria that have active membrane potential, to measure mitochondrial membrane potential. Data shown in are in average ± sem, n=6 biologically independent samples/group.

Fig 3: Biochemical characterization of OPA interactions with HIGD2A and COX5A.(a-f) Competitive gel-based ABPP analysis of OPA, 6-epi-OPA, and anhydro-6-epi-OPA binding to HIG2DA and COX5A. Pure proteins were pre-incubated with either DMSO, OPA, 6-epi-OPA, or anhydro-6-epi-OPA (30 min) prior to labeling of protein with either a cysteine-reactive rhodamine-functionalized iodoacetamide (IA-rhodamine) probe or lysine reactive rhodamine-functionalized NHS-ester probe (NHS-ester-rhodamine) probe. HIGD2A was labeled with 0.5 μM IA-rhodamine and COX5A labeled with 10 μM NHS-ester rhodamine. (g) Structure of alkyne-functionalized OPA probe (OPA-alkyne). (h, i) OPA-alkyne labeling of COX5A and HIG2DA. HIG2DA and COX5A pure protein (0.2 μg) were pre-incubated with DMSO vehicle or OPA (50 μM) for 30 min prior to labeling with OPA-alkyne probe for 60 min. (j, k) OPA engagement of HIG2DA and COX5A in NCI-H1703 cells. NCI-H1703 cells were treated with DMSO vehicle or OPA-alkyne (5 μM) for 1 h after which probe-modified proteins from cell lysate were appended to an azide-functionalized biotin enrichment handle for avidin enrichment and elution and blotted for HIGD2A or COX5A with GAPDH as the negative control GAPDH. Both input and pulldown protein levels are shown. Gels shown in (a-f, h,i,j,k) are representative of n=3 biologically independent replicates/group.