2 6 dichlorophenolindophenol sodium salt dcip

In Supplementary Tables 5 and 6, endpoint assays were performed with 10 nM recombinant human DHODH (#ENZ-642, Prospecbio). The reaction mixture consisted of 624.6 µM DL-dihydroorotic acid, 66.4 µM coenzyme Q₁₀, 66.4 µM 2,6-dichlorophenolindophenol sodium salt (DCIP) (all reagents purchased from Sigma-Aldrich) in enzyme buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 150 mM KCl). Loss in absorbance by DCIP was measured at 595 nm after incubation at RT for 60 min.
In Fig. 5a enzyme assays were optimized and performed with 6 nM recombinant human DHODH prepared as described^{13 (link)}. The reaction mixture for these kinetic assays consisted of 1 mM DL-dihydroorotic acid, 100 µM 3,4-dimethoxy-5-methyl-p-benzoquinone (#D9150, Sigma-Aldrich), and 100 µM DCIP in enzyme buffer. A stock solution of 20 mM DCIP was prepared in enzyme buffer and filtered through filter paper (20–25 μm pore size) just before use. Loss in absorbance by DCIP was measured at 595 nm at RT in a stepped time course (8 × 2 min, 8 × 3 min, 6 × 5 min). The observed decrease in absorbance over time was linear between 8 and 26 min. Therefore, for each concentration of inhibitor tested, a value for DHODH’s V_max was estimated by linear regression within this time frame. The IC₅₀ is defined as the concentration of inhibitor that gives V_max ([I]) = V_max (DMSO)/2.

Mitochondria isolated from cultured cells were used for the detection of complex II enzyme activity 42 (link). The activity of succinate-coenzyme Q reductase (SQR) and succinate dehydrogenase (SDH) was tested as previously described 43 (link). For SQR activity, mitochondria isolated from 2 × 10⁷ cells were incubated with potassium phosphate buffer (KPi buffer) containing succinate (20 mM, Sigma-Aldrich), antimycin (32 μM, Sigma-Aldrich), rotenone (12 μM), NaN₃ (5 mM, Sangon Biotech), 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone (DB, 20 μM, Sigma-Aldrich), and 2,6-Dichlorophenolindophenolsodium salt (DCIP, 50 μM, Sigma-Aldrich). Absorbance was monitored at 600 nm. For SDH activity, mitochondria were incubated with Tris-HCl buffer containing succinate (10 mM), 60 μg/mL methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma-Aldrich), 120 μg/mL PMS, antimycin (32 μM), rotenone (12 μM), and NaN₃ (5 mM). Absorbance was monitored at 570 nm. The data were normalized with the mitochondrial protein concentration.