Mitogreen

After 48 h of intervention, the changes in ROS levels of MC3TE-E1 cells following the intervention were evaluated using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma, St. Louis, MO) staining and MitoSOX™ Red mitochondrial superoxide indicator (MitoSOX™; Sigma, St. Louis, MO) staining.
To confirm the changes in mitochondrial membrane potential, we use co-stained with tetramethylrhodamine methyl ester (TMRM, 100 nM, Life Technologies) and Mitotracker Green (Mitogreen, 100 nM, Life Technologies) to observe and evaluate the state of the mitochondrial oxidative stress in treated cells. After 48 h of intervention, TMRM and Mitogreen staining were performed as previously described [30 (link)]. The results of TMRM (excitation wavelengths = 543 nm) and Mitogreen (excitation wavelengths = 488 nm) were captured using Laser confocal microscope. The quantification of mitochondrial membrane potential was determined using Image J software.
Immunofluorescence staining was used to quantitatively detect and evaluate the expression of SIRT1 and SOD2 in MC3T3-E1 after different interventions to understand the changes of cellular oxidative stress as stated above. The levels of Malondialdehyde (MDA) and total SOD activity in the cells were measured by a malondialdehyde Detection Assay kit (APPLYGEN) and Superoxide Dismutase Activity Assay Kit (Abcam), respectively.

Dulbecco’s Modified Eagle Medium (DMEM) (#11995065), Fetal bovine serum (FBS) (#16000-044), MitoSOX Red (#M36008), Mitogreen (#M7514), and TMRM (#T668) were purchased from Life Technologies (Grand Island, NY, USA). TUNEL assay kit was from Roche (Mannheim, Germany). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from BD Bioscience (NJ, USA). Fluo-4-AM, ATP assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Glucose oxidase (GO) (#G2133), N-acetylcysteine (NAC) (#A7250), Cyclosporine A (CsA) (#SML1018) and Lithium chloride (LiCl) (#L9650) were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 (#9901) was from Cell Signaling Technology (Beverly, MA, USA). Antibodies against phosphorylated Akt (Ser473) (p-Akt) (#4060 S), Akt (#2920 S), phosphorylated GSK3β (Ser9) (p-GSK3β) (#9336 S), GSK3β (#9832 S), Bax (#2772 S), Bcl2 (#3498 S), Caspase-3 (#9662 S), and β-actin (#3700) were obtained from Cell Signaling Technology (Beverly, MA, USA), and antibody CypD (ab110324) was obtained from Abcam (USA). Chamber slides, goat anti-rabbit (#656120) and anti-mouse (#626520) secondary antibodies, and DAPI (#P36931) were obtained from Invitrogen (Carlsbad, CA, USA)

To assess the intracellular localization of LA, it was labeled with the fluorescent reagent 9-anthryldiazomethane (ADAM; Funakoshi, Tokyo, Japan). ADAM reacts with fatty acids to form esters that emit fluorescence with strong intensity. Methanol (1 mL) was added to ADAM (1 mg) to prepare a 0.1% ADAM reaction solution. The reaction solution (100 μL) and LA (100 μL) were mixed in the dark at room temperature for 2 h. CT26 cells were then treated with labeled LA (35 μg/mL) and Mitogreen (100 nM)(Molecular Probes, Eugene, OR, USA) for visualizing mitochondria. Images were observed using a fluorescence microscope (BZ-X700, KEYENCE, Osaka, Japan). Fluorescence intensities of the labeled-LA solution (35 μg/mL) and the whole cell lysate and nuclear fraction of labeled LA-treated CT26 cells were measured by a fluorescence spectrophotometer (F-2700, Hitachi High-Tech GLOBAL, Tokyo, Japan).

DHE, DAPI, CellROX, MitoGreen, MitoRed, MitoSOX, Hochest 33342, and chloromethyl‐2',7'‐dichlorodihydrofluorescein diacetate (CM‐H₂DCFDA) were purchased from Molecular Probes (Eugene, OR). Glucose, BSA, trypsin, palmitate, NADPH, and histopaque‐1077 were purchased from Sigma (St. Louis, MO). Lucigenin, and collagenase P were from Roche (Switzerland). GHTT was purified as described previously (Almeida et al, 2011 (link); Kuo et al, 2017 (link)). Recombinant proteins, 6×His‐Pdia4, Gst‐p22^phox and Gst‐Ndufs3 were purchased from Enzo (Farmingdale, NY). Gst‐TecSH3, containing a SH3 domain of Tec, was produced as published (Kuo et al, 2017 (link)). The antibodies used in this study were purchased (Appendix Table S3). 293T cells (CRL‐3216), Min6 cells (Yagi et al, 1995 (link)), and pancreatic islets were grown in complete DMEM medium (Thermo Fisher, Waltham, MA) containing 10% and 20% fetal bovine serum (FBS), respectively, and 3.3 mM glucose unless indicated otherwise. Human islets were purchased from Lonza (Switzerland) and handled according to the protocol of the Academia Sinica Institutional Review Board (AS‐IRB01‐14015). Informed consent was obtained from all human subjects and the experiments conformed to the principles of the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.