2 7 dichlorofluorescein diacetate h2dcfda

The human ARPE-19 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 media (DMEM/F12), fetal bovine serum (FBS), penicillin/streptomycin, and 2,7-dichlorofluorescein diacetate (H₂DCF-DA) were purchased from Thermo Fisher Scientific (Wilmington, DE, USA). Hydrogen peroxide, dihydroethidium (DHE), JC-10 assay kit, N-acetyl-cysteine (NAC), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma-Aldrich (St. Louis, MO, USA, owned by Merck KGA). TPP-Niacin was chemically synthesized according to a previous report and patent application, and niacin was used as the reference control [12 (link), 14 (link), 22 (link), 23 ]. The Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays were purchased from Dojindo Molecular Technologies (Japan). The kits used to determine the superoxide dismutase 1 and 2 (SOD1 and SOD2), catalase and glutathione peroxidase activities, and tetramethylrhodamine ethyl ester (TMRE) reagent were obtained from Abcam (UK).

All of the chemicals used in the present study were analytical grade reagents from various sources. Phosphate-buffered saline (PBS), formaldehyde acetic acid and ammonium hydroxide, aminoguanidine bicarbonate (AG), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and FPS-ZM1 were purchased from Merck Spa (Milan, Italy). Laemmli buffer and 2′–7′-dichlorofluorescein-diacetate (H₂DCF-DA) were purchased by Thermo Fisher Scientific (Milan, Italy), Roti-Block from Prodotti Gianni (Milan, Italy), and bicinchoninic acid (BCA) kit from Thermo Fisher Scientific (Milan, Italy).

Hexafluoro butyric acid (PFBA; 98% purity), methanol (LC-MS grade), sulfuric acid, titanium tetrachloride, hydrogen peroxide (3%), potassium phosphate monobasic, and dibasic (for phosphate buffer preparation), quercetin and gallic acid were purchased from Sigma-Aldrich (Allentown, PA, USA). Bovine serum albumin (BSA), Coomassie reagent, 2′, 7′ -dichlorofluorescein diacetate (H₂DCFDA), and SOD kit were purchased from Thermo Fisher Scientific (Allenstown, PA, USA). Nanopure water (Thermo Fisher Scientific) was used to prepare all solutions.

C3G (purity ≥99%, Fig. 1), L-glutamic acid, Dulbecco’s modified Eagle medium (DMEM), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against calpain, caspase12, CHOP, ERK, p-ERK (Thr202/Tyr204), β-actin and horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and Nrf2 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Bio basic (Toronto, Canada). Dimethylsulfoxide (DMSO) was purchased from Merck (Darmstadt, Germany). Penicillin-streptomycin solution was obtained from Corning Inc. (Corning, NY, USA). Annexin V FITC/PI kit was purchased from Biolegend (San Diego, CA, USA). The reagent kit for lactate dehydrogenase (LDH) was bought from Promega’s CytoTox 96™ (Madison, WI, USA) and 2′,7′- dichlorofluorescein diacetate (H₂DCF-DA) was obtained from Life technology (Carlsbad, CA, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA).
Fig. 1

Chemical structure of C3G

Isolated PBNs were stained with 2 μmol of calcium aceto-methyl ester (Life Technologies, Eugene, OR, USA) for the migration assay and 2′,7′-dichlorofluorescein diacetate (H2DCFDA) (Life Technologies) for the ROS assay for 30 min at 37 °C, after which the cells were washed once with 1× HBSS. For the migration assay, the cells were pretreated with anti-S100A9 for 30 min and then seeded on the upper chamber with a pore size of 3.0 μm (Neuro Probe, Gaithersburg, MD, USA). Phenol red-free RPMI medium containing S100A9 was added to the lower chamber, and the Transwell plate was incubated for 2 h at 37 °C. For the ROS assay, the cells were pretreated with anti-S100A9 for 30 min in phenol red-free RPMI and then treated with S100A9 for 30 min. The signal was measured at excitation and emission wavelengths of 480 and 520 nm, respectively, with a fluorescence microplate reader (Synergy HT; BioTek Instrument).

Unless specified, all reagents were obtained from Sigma and all the antibodies were from Santa Cruz Biotechnology, except for anti-HIF-1α (Becton Dickinson), anti-PKM2 and anti-Src-Tyr416 (Cell Signaling Technology) and anti phospho-Tyr (clone 4G10) (Millipore). HRP-conjugated streptavidin was from Pierce. HIF-1-siRNA and DEC1-siRNA were from Santa Cruz Biotechnology. N-(biotinoyl)-N-(iodoacetyl)ethylenediamine (BIAM) and 2′-7′-dichlorofluoresceindiacetate (H₂-DCF-DA) were from Molecular Probe. [U-¹⁴C] lactate and [U-¹⁴C] glucose were from Perkin Elmer. All the kits used to perform miRNA extraction and quantitative reverse transcriptase PCR were from Qiagen. Lipofectamine 2000 was from Invitrogen. Metformin was obtained by Sigma. The chemical synthesis of the PKM2 activator DASA-58 was kindly performed by Dr. Richichi of the Department of Chemistry (University of Florence). The mitochondrial antioxidant MitoTEMPO was from Santa Cruz Biotechnology. The MCT1 inhibitor, AR-C155858, was from Tocris Bioscience.