Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp

(R)‐salbutamol (>99% purity, 99.85% ee) and (S)‐salbutamol (>99% purity, 92.73% ee) were provided by Dongguan Key‐Pharma Biomedical Co., Ltd. LPS (Escherichia coli O111:B4), ICI‐ 118551 hydrochloride, fluorescent probes 3‐Amino,4‐aminomethyl‐2′,7′‐difluorescein diacetate (DAF‐FM DA) and 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) were bought from Sigma Chemical Co. The kits for cDNA synthesis, BCA protein assay, cell culture reagents and SYBR Green Supermix were from by Life Technologies Inc (Gibco). Methanol and acetonitrile were acquired from Fisher Chemical. Phycoerythrin (PE)‐conjugated anti‐mouse F4/80 (123110), fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse CD206 (141704) and allophycocyanin (APC)‐conjugated anti‐mouse CD11c (117310) were procured from BioLegend. β‐actin antibody (# BF01980) was obtained from Affinity Biosciences. Inducible nitric oxide synthase (iNOS) mouse antibody (2982S) was obtained from Cell Signaling Technology. The enzyme immunoassay kits for MCP‐1, IL‐1β and TNF‐α were manufactured by Neobioscience. Beyotime Institute of Biotechnology supplied the Cell Counting Kit‐8 (CCK‐8). Rotenone/antimycin A, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin came from Seahorse Bioscience (Agilent Technologies, Inc).

OCR and ECAR were assessed using a Seahorse XF-96 extracellular flux analyzer (Seahorse Bioscience). DLBCL cells were pooled, carefully counted and plated (1 × 10⁵ cells/well) in assay medium onto Seahorse cell plates coated with Cell-Tak (Corning, Inc.). For OCR, assay medium was supplied with XF Base Medium with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (Sigma-Aldrich). Baseline OCR was defined as the OCR readings before 1 μM oligomycin (Seahorse Bioscience) injection. SRC was defined as the difference between baseline OCR and maximal OCR after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.5 μM, Seahorse Bioscience) injection to uncouple oxidative phosphorylation and electron transport. For ECAR, assay medium was supplied with XF Base Medium with 2 mM glutamine. Glycolysis was defined as the ECAR reached by a given cell after the addition of saturating amounts of glucose. Glycolytic reserve was defined as the difference in ECAR between the glucose and 1 μM oligomycin injections.

Oxygen consumption and extracellular acidification rates in PEL cells were measured using the Seahorse XF96 instrument (Seahorse Biosciences, North Billerica, MA) according to the manufacturer's protocols. 24 hours after treatment, cells were seeded in a poly-lysine coated XF96 microplate at the density of 150000 cells per well, for 60 minutes in a 37°C non-CO2 incubator, while sensor cartridges were calibrated prior to the start of an assay. After adhesion, medium was changed with 175 μl unbuffered XF assay media at pH 7.4 supplemented with 5.5 mM glucose (Sigma), 1 mM sodium pyruvate and 1 mM Glutamine. Respiration was measured in four blocks of three for 3 min. The first block measured the basal respiration rate. Next 2 μM oligomycin was added to inhibit complex V (second block). Then 0.3 μM carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP, Seahorse Biosciences, North Billerica, MA) was added to uncouple respiration (third block). Finally, 1 μM antimycin A and 1 μM Rotenone was added to inhibit complex III (forth block). All reagents are from Seahorse Biosciences, North Billerica, MA. Immediately after finishing the measurements, cells were washed with phosphate-buffered saline, fixed with 4% paraphormaldehyde and stained with 0.1% crystal violet (1 mol/l acetic acid) and absorbance at 595 nm was measured as index of cell amount.

Ten thousand melanoma cells were plated in Seahorse XFp cell culture miniplates for 16-18 hours before mitochondrial function assays. The cells were subsequently treated with PET-16 for 24 hours, and subjected to the Seahorse XF Cell Mito Stress Test, according to manufacturer's protocol. Briefly, cell medium was replaced with Seahorse XF Base Medium (supplemented with 100 mM Pyruvate, 200 mM Glutamine, and 2.5 M Glucose) and incubated in a non-CO₂ incubator for one hour before the start of the assay. Basal OCR was measured using the Seahorse XFp Extracellular Flux analyzer. Measurements were performed after injection of three compounds affecting bioenergetics: 1 μM oligomycin (Seahorse Bioscience, North Billerica, MA), 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Seahorse Bioscience) and 0.5 μM Rotenone/Antimycin A (Seahorse Bioscience). Upon completion of the Seahorse XFp Flux analysis, cells were lysed to calculate the protein concentration. The results were normalized to protein abundance in corresponding wells. Data are representative of three biological replicates.

Dimethyl sulfoxide (DMSO), 2-deoxy-D-glucose (2-DG), D-(+)-glucose, D-(+)-galactose, sodium dichloroacetate (DCA), sodium pyruvate, sodium L-lactate, H-89 dihydrochloride hydrate (H-89), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was obtained from Seahorse Bioscience (Billerica, MA, USA). FITC Anti-human CD14 monoclonal (M5-E2) and A647 anti-human CD163 monoclonal (RM3/1) were purchased from Sony Biotechnology Inc. (San Jose, California, USA). PE anti-human CD11b monoclonal (ICRF44) was obtained from BD Bioscience (Vianen, The Netherlands). Anti-human PDH-E1a (pSer²⁹³) and anti-human PDH-E1a (pSer³⁰⁰) polyclonal antibodies were purchased from Merck Millipore (Amsterdam, The Netherlands). Anti-human vinculin monoclonal (hVIN-1) was purchased from Sigma-Aldrich and anti-human PDH monoclonal (9H9AF5) from Thermo Fisher Scientific (Landsmeer, The Netherlands). Secondary stabilized peroxidase conjugated antibodies goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) were purchased from Thermo Fisher Scientific.

Pretreated HCMEC/D3s were seeded in Seahorse XF96 plates (Agilent, China) with the amount of 20,000 cells per well. Cells were incubated with nonglucose medium for 1 h before undergoing the glycolysis stress evaluation (Seahorse XF96 analyzers, Agilent, China) by detecting the extracellular acidification rate (ECAR) of cells. Detection was carried out per 5 min before and after sequentially adding with glucose, oligomycin and 2‐dexoy‐d‐glucose (2‐DG) (Agilent, China). Next, cells were incubated with normal medium for 1 h before the mitochondrial stress evaluation by detecting the oxygen consumption rate (OCR) of cells. And detection was carried out per 5 min before and after sequentially adding with oligomycin, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP) and rotenone/antimycin A (Agilent, China). Wave software and Graphpad were employed to process and analyze the data obtained.²⁵