Acridine orange ao

Acridine Orange (AO; Invitrogen, Waltham, MA, USA) was used as a vital dye that penetrates cells. It stains nucleic acids and simultaneously, having pH-sensing properties, visualizes acidic intracellular structures including autophagosomes. The experiments were carried out on 10–12-day old Arabidopsis seedlings. The experimental groups of seedlings were incubated in phytotoxin solutions containing 100 mM sorbitol and 0.1% DMSO, supplemented with either 10 µg/mL STA or 50 µg/mL HBI. These concentrations of phytotoxins are known as non-lethal but inhibiting root growth [24 (link)]. After incubation in toxin-free control or in toxin-supplemented experimental solutions, the seedlings were transferred to an aqueous solution of 25 µM AO for 10 min, then washed for 1 min in a buffer of 100 mM sorbitol, 2 mM Tris, 1 mM MES, 0.1 mM KCl, 0.1 mM CaCl₂, pH 6.0 and analyzed under a confocal microscope LSM 780 (Carl Zeiss, Oberkochen, Germany) with a Plan-Apochromat objective 40×/1.4 Oil DIC. AO fluorescence was excited by a 488 nm argon laser; the fluorescence signal was detected in two channels, red and green (615–660 nm and 530–540 nm, respectively). To validate the method for assessing the pH of the apoplast using the AO fluorescent dye, experiments were performed in which the plasmalemma H⁺-ATPase inhibitor sodium orthovanadate (Na₃VO₄, Sigma-Aldrich) was used as a positive control.

Lysosome permeability was analyzed by fluorescence microscopy using the Operetta High Content Imaging System from PerkinElmer (Waltham, MA). A2780 cells were plated in Cell Carrier-96 plate from PerkinElmer (Waltham, MA) at a density of 10,000 cells per well. Forty-eight hours after plating, the cells were treated with ZnPP or SnPP or pre-treated with Brefeldin A/Monensin (21.1 μM / 4 μM) cocktail for 4 hours followed by treatment with ZnPP. The medium was then replaced with fresh medium containing 2.5 μM Acridine Orange (AO, Invitrogen, Carlsbad, CA). After 30 minutes incubation the cells were washed three times with HBSS and viewed under the Operetta. Lysosome permeability was measured using the AO staining [14 (link)]. AO was detected by excitation at 500 nm, emission at 526 nm for red, and excitation at 460 nm, emission at 650 nm for green.

DHA was purchased from Aladdin Co. Ltd. (Shanghai, China). 1, 2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), BSO and FeSO₄•7H₂O were obtained from Sigma-Aldrich (St. Louis, MO, USA). Deferiprone (DEF) was purchased from Meyer Chemical Technology Co. Ltd (Shanghai, China). ROS Detection Kit, Glutathione Assay Kit, Annexin V-FITC/Propidium Iodide (PI) Cell Apoptosis Detection Kit, dihydroethidium (DHE), and Protein Extraction Kit were obtained from KeyGen Biotech. Co. Ltd. (Nanjing, China). BCA Protein Assay Kit was purchased from Beyo-time Institute of Biotechnology (Shanghai, China). The primary antibodies and secondary antibody against TfR and GAPDH were acquired from Affinity Biosciences (Changzhou, China). Fluorescein isothiocyanate (FITC), CellROX, LysoTracker Red, MitoTracker Red, Hoechst 33342, acridine orange (AO) and LIVE/DEAD™ Fixable Green Dead were obtained from Invitrogen (ThermoFisher Scientific, USA). Iron Colorimetric Assay Kit was purchased from BioVision (San Francisco, USA). 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol) 2000-transferrin (DSPE-PEG₂₀₀₀-Tf) was synthesized by Xi-An Ruixi Biological Technology Co., Ltd. (Xi-An, China). Ultrapure water was prepared using a Millipore Simplicity System (Millipore, Bedford, USA).