Sodium fluoride

All reagents were of the highest commercially available grade. DHMBA was synthesized, as previously described [17 (link)]. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye and the JC-1 fluorescent probe were purchased from Dojindo (Kumamoto, Japan). Antibodies against PI3K, phospho-PI3K Tyr⁴⁵⁸, Akt, phospho-Akt Ser⁴⁷³, mTOR, phospho-mTOR Ser²⁴⁴⁸, p70S6K, phospho-p70S6K Thr³⁸⁹, β-actin, apoptosis-inducing factor (AIF), COX IV, and goat anti-rabbit IgG antibody conjugated with horseradish peroxidase, were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against HO-1 and NQO-1 were purchased from Abcam (Cambridge, UK). Primary and secondary antibodies were diluted in tris-buffered saline (pH 7.4) with Tween20 (0.05%, v/v) to 1:1000 and 1:5000, respectively. Following reagents were purchased from Fuji Film Wako Pure Chemical Co. (Osaka, Japan): Trolox, Dimethyl sulfoxide (DMSO), Edaravone, Sodium fluoride, Sodium orthovanadate (V), and Bovine serum albumin fraction V (BSA). Following chemicals were purchased from Merck Inc. (Darmstadt, Germany): Deferoxamine mesylate (DFX), Ebselen, Protease inhibitor cocktail, and 2-Mercaputoethanol (2-ME).

The volatile standards used in this study are shown in Additional File 4: Table S1. First, 2,4-dichloroaniline (99.5% purity) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) for the reagents. However, while n-heptyl β-D-glucopyranoside (98% purity) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), dichloromethane (99.5% purity), liquid chromatography-mass spectrometry-grade methanol, citric acid, sodium phosphates, sodium fluoride (NaF), ascorbic acid, sodium hydroxide (NaOH) and 3-octanol (97% purity) were obtained from Wako Co. Ltd. (Osaka, Japan).

Western blot analysis was performed on the basis of a previous report [65 (link)]. Briefly, HPDL cells were lysed in RIPA buffer (Millipore, MA, USA) containing a protease inhibitor cocktail (Roche, IN, USA), 1 mM sodium orthovanadate (Sigma-Aldrich), 1 mM Sodium fluoride, and 10 mM β-glycerophosphoric acid (Wako). Protein concentrations of the lysates were quantified by the Bradford Assay (Bio-Rad, CA, USA). Lysates were denatured in 5× Laemmli buffer containing 2-mercaptoethanol by boiling for 10 min at 95°C. After cooling, the proteins were separated by SDS-PAGE under reducing conditions and transferred to a PVDF membrane (GE Healthcare, IN, USA). Membranes were blocked with 5% dry skim milk for 1 h and then probed with the following primary antibodies: mouse anti-human p53, rabbit anti-human p16, goat anti-human CTGF (Santa Cruz, TX, USA), rabbit anti-human p21, mouse anti-human Rb, rabbit anti-human SIRT1 (CST), and mouse anti-human β-actin (Sigma-Aldrich). After washing, membranes were incubated with secondary antibodies, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG or donkey anti-goat IgG (CST), and visualized with ECL prime Western Blotting Detection Reagents (GE Healthcare).