br RIKEN NPDepo http www npd riken jp npd
RIKEN NPDepo (http://www.npd.riken.jp/npd/en/) has collected our own and commercially available natural products and their deri-vatives. Unantimycin A, SW-163A, and opantimycin A were isolated by ourselves from the fermentation broth of the actinomycete strain RK88-1355 [32, 33]. NPL40330 was purchased from AnalytiCon Discovery GmbH (Potsdam, Germany). Most of the standard inhibitors for the metabolic machinery, as well as the chemical library used for screening, were obtained from NPDepo. The purities and structures of the hit compounds were confirmed by liquid chromatography-mass spectro-metry analysis (Fig. S1). Potassium cyanide (KCN) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). L(+)-ascorbic acid, DL-malic acid, pyruvic acid, sodium azide, and succinic Calcein acetoxymethyl ester were purchased from Nacalai Tesque (Kyoto, Japan). Adenosine diphosphate (ADP), etomoxir, carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), malonic acid, sodium pyruvate, N,N,N′,N′-tetramethyl-p-phe-nylenediamine (TMPD), and UK5099 were purchased from Sigma-Al-drich (St. Louis, MO, USA). Duroquinol, GlutaMAX, and digitonin were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Thermo Fisher Scientific (Waltham, MA, USA), and Wako Pure Che-mical Industries, Ltd. (Osaka, Japan), respectively.
2.2. Cell culture and cytotoxicity test
The in vitro cytotoxicity assay method against human cervix epi-dermoid carcinoma cell line HeLa was described in the previous report . Briefly, cells were seeded at 4 × 103 cells/well into a 96-well plate and exposed to the test compounds for 48 h. Cell proliferation was determined using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Note that this cell line was verified to be free of mycoplasma contamination by Hoechst staining.
2.3. Assessment of metabolic phenotypes
the Seahorse XFe96 microplates (Agilent Technologies) and pre-incubated in DMEM-based assay medium (Agilent Technologies) sup-plemented with 25 mM glucose, 4 mM GlutaMAX, and 1 mM sodium pyruvate for 1 h at 37 °C in a CO2-free incubator. After baseline mea-surements were taken, a test sample was injected from port A. Changes in OCR/ECAR values were simultaneously recorded in triplicate every 6 min until ten sets of values were obtained. The average value was calculated in each instance. Next, measurements were taken using the Seahorse XF Cell Mito Stress Test kit (Agilent Technologies) following sequential injections of oligomycin A (1 μM) from port B, FCCP (0.125 μM) from port C, and antimycin A (1 μM) plus rotenone (1 μM) from port D. Percentage changes in OCR and ECAR were calculated with respect to basal OCR/ECAR values.
2.4. Two-dimensional electrophoresis-based proteomic analysis
ChemProteoBase profiling was performed as previously described . Briefly, HeLa cells were treated with unantimycin A (48 μM) or NPL40330 (5 μM) for 18 h. For the biological replicate, cell lysates were prepared in triplicate against each compound. Proteome analysis of cell lysates was then performed using a 2-D DIGE system (GE Healthcare, Little Chalfont, UK), after which images of the gels were analyzed using Progenesis SameSpots (Nonlinear Dynamics, Newcastle upon Tyne, UK). Out of > 1000 spots that were detected on each gel, 296 diﬀerent spots were found in common between gels of reference-compound-treated cells and selected. Next, 273 of the selected spots were identi-fied by matrix-assisted laser desorption ionization-time of flight mass spectrometry (ultraflex II; Bruker Daltonics, Billerica, MA, USA) or li-quid chromatography-tandem mass spectrometry (Q Exactive, Thermo Fisher Scientific) using Mascot search (Matrix Science, Boston, MA, USA) (Table S1). Each spot in the list was identified at least twice as the same protein. The log ratio values of spot expression between com-pound-treated cells and control cells were plotted as a waterfall plot. The 296 bars were ordered in decreasing order of the log ratio of each spot. The bars corresponding to the protein spot that includes a key-word in the Comment (CC), Database cross-Reference (DR), and the KeyWord (KW) lines of UniProt database  (http://www.uniprot. org) were designated as black bars. Each score is the total sum of the log ratio selected by a keyword.
2.5. In vitro reconstitution assay for mitochondrial electron transport chain (ETC)
In vitro reconstitution assay for mitochondrial ETC was carried out by following an established method  with some modifications in the concentrations of digitonin and the substrates. HeLa cells (1 × 104 cells/well in 80 μl DMEM) seeded in microplates were incubated overnight at 37 °C under 5% CO2. The culture medium was replaced with MAS buﬀer (220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES-KOH, and 1 mM ethylene glycol tetraacetic acid, pH 7.2), after which the cells were incubated for 15 min at 37 °C in a CO2-free incubator. Next, three baseline measurements were taken using the Seahorse XFe96 analyzer, after which 25 μl of MAS buﬀer containing digitonin, 10 mM sodium succinate, 1 μM rotenone, and 1 mM ADP was injected from port A to start the reaction. This was followed by sequential treatments with oligomycin A (1 μM) from port B and antimycin A (1 μM) or sodium azide (20 mM) from port C. Suc-cinate plus rotenone was replaced with malate (1 mM) plus pyruvate (10 mM) for the assessment of complex I, duroquinol (0.5 mM) for the assessment of complex III, or TMPD (0.5 mM) plus sodium ascorbate (2 mM) for the assessment of complex IV. When indicated, typical re-spiration inhibitors or test samples were co-injected with ADP, sub-strates, or digitonin.