Glutathione (gsh)

The following materials were purchased from Fisher Scientific (Pittsburgh, PA, USA): hydrochloric acid (HCl), ammonium hydroxide, sodium hydroxide (NaOH), glutathione (GSH), RPMI-1640 medium, fetal bovine serum (FBS), and TrypLE. Sodium carbonate was purchased from EMD Millipore (Billerica, MA, USA). Bis[3-(triethoxysilyl)propyl] disulfide (BTEPDS) was obtained from Gelest (Morrisville, PA, USA). Tetraethyl orthosilicate (TEOS), triethylamine (TEA), and hexadecyltrimethylammonium bromide (CTAB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethanol 95% and absolute ethanol were purchased from Decon Labs (King of Prussia, PA, USA). RAW 264.7 macrophage cell line was received from ATCC (Manassas, VA, USA). The cell counting kit-8 (CCK-8) cytotoxicity assay was purchased from Dojindo (Rockville, MD, USA). Simulated body fluid (SBF) pH 7.4 was purchased from Biochemazone (Waterloo, ON, Canada). SBF was used due to the ionic strength and composition closely resembling physiological conditions compared to other aqueous mediums such as phosphate buffer saline. The pH of SBF with 10 mM GSH was adjusted to pH 7.4 with NaOH.

For pharmacological feeding experiments, third instar larvae were grown in fly flood containing buffer (0.01% DMSO), 1 µM BFA1 (Cayman Chemical), 25 mM H₂O₂ (Fisher), 5 mM DA (Fisher), or 0.5 mM GSH (Fisher) (Table S1) dissolved in 0.01%DMSO for 3 h prior to dissection and whole-mounting of larval axons for in vivo simultaneous dual-view imaging of either MitoTimer (568/488 nm), Mito-roGFP2-ORP1 (405/488 nm) or Mito-roGFP2-GRX1 (405/488 nm) as described above. For Mdivi1 feeding, larvae were fed food containing 10 µM Mdivi-1 (Fisher) dissolved in 0.01% DMSO, or food containing 0.01% DMSO alone, for 8 h prior to dissection and subsequent in vivo simultaneous dual-view imaging of MitoTimer or immunohistochemistry with cytochrome C (Abcam, 1:500) as described above. Note that feeding larvae at high concentrations of Mdivi-1 (≥500 µM) induced lethality; feeding heterozygous DRP1 mutant larvae Mdivi-1 at 10 µM (8 h) caused elongated mitochondria (Fig. 5A). For stress conditions, larvae expressing MitoTimer were encapsulated in a tube containing dissection buffer and placed in either a bath of room temperature water (22 °C), cold water (4 °C), warm water (37 °C), adhered to a low speed vortex, or treated with a global bath of 500 nM BFA1 (Cayman Chemicals) for 1 h prior to dissection and in vivo imaging.

BITC was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) to create a 10 mM stock solution. A glutathione (GSH) (G4705, Sigma-Aldrich Inc., Darmstadt, Germany) stock solution was prepared by dissolving GSH in HPLC-grade water (Fisher Scientific Korea Ltd., Gangnam-gu, Seoul, Korea), and this stock solution was diluted in the culture medium before treatment of cells to obtain the final working solution. RPMI-1640 medium was purchased from Welgene Inc. (Daegu, Korea), and fetal bovine serum (FBS) was purchased from Corning Inc. (Corning, NY, USA). Antibodies against cleaved Caspase-3 (c-Cas-3) (9664), Caspase-8 (9746), Caspase-9 (9508), poly (ADP-ribose) polymerase (PARP) (9532), X-linked inhibitor of apoptosis protein (XIAP) (14334), DR4 (42533) and DR5 (8074) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibodies were bought from Enzo Life Sciences (Farmingdale, NY, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-32233), Bcl-2 (sc-492), Bax (sc-493) and Cyt c (sc-13156) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).