8 well chamber

Human OA chondrocytes or C28I2 cells were seeded on 8-well chamber (Thermo Fisher, 154461) and loaded with 5 µM CoroNa Green (Invitrogen, C36676) or Fluo-8 (Abcam, ab112129) in Hanks’ Balanced Salt Solution for 45 min at 37 °C in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264 with or without 0.5 µM KB-R7943 (Tocris, 1244). CoroNa Green and Fluo-8 were excited at 488 nm and fluorescence images (525–530 nm) were acquired with 25x water-dipping objective on Zeiss 880 confocal microscope every 2 s during the experiment. Na⁺ and Ca²⁺ transients in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was expressed as the ratio of cytosolic fluorescence and initial intensity (F/F₀).

Glutamine and glutamate intracellular concentrations were calculated using the Glutamine/Glutamate-Glo^TM Assay (Promega, Wisconsin, USA) following the manufacturer’s protocol. Mitochondrial morphology analysis was performed as described in ref. ⁴² Briefly, the live cell fraction was isolated from T cells stimulated with plate-bound anti-CD3/CD28 by using lymphocyte separation medium (Corning, Virginia, USA). These cells were then stained in prewarmed serum-free medium containing MitoTracker Green at a concentration of 1 μM for 40 min at 37 °C. The cells were then washed twice with PBS to remove any residual MitoTracker and resuspended in 100 μl of PBS with a 1:1000 dilution of DAPI. The cells were then washed and plated in an 8-well chamber (Nunc, New York, USA) to form a monolayer for microscopic analysis. The samples were then analyzed on an IX83 fluorescence microscope (Olympus, Tokyo, Japan). The images were collected and analyzed on cellSens imaging software (Olympus, Tokyo, Japan).

Live cell imaging was performed on a Zeiss 800 Confocal Laser Scanning Microscope (Zeiss, Germany) at the La Trobe Bioimaging Platform using 63× oil immersion at 37 °C and 5% CO₂. All cells were imaged in an 8-well chamber (Nunc, Denmark) and prepared using 1% BSA RPMI. A549 cells (2 × 10⁴ cells/well) were seeded the day before imaging to allow for adherence. Jurkat cells (1.5 × 10⁵ cells/well) were immobilized using 10% poly-L-lysine coated chambers. In all experiments, GS-1 was prepared at 2.7 mM for A549 s or 2 mM for Jurkats. Stains including MitoTracker Red (Thermo Fisher Scientific, MA) and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY) (0.1 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA), were prepared and added to cells prior to imaging. In certain experiments cells were treated with inhibitors including 50 µM Grassofermata (inhibitor of FATP2) (Sapphire Bioscience, NSW, AUS) or Diglyceride acyltransferase inhibitor 1 (DGATi1; 2 mM) and Diglyceride acyltransferase inhibitor (DGATi2; 10 mM) (kind gifts from Professor Karla Helbig; collectively referred to as DGATi), inhibitors of lipid droplet biogenesis, prior to imaging. Cells were imaged at time points stated in figure legends. Images were analyzed using Zen Image Analysis software (Zen Blue Edition version 10.1.19043, Jena, Germany).

For all imaging performed in this paper, cells were seeded in an 8-well chamber (Nunc, Roskilde, Sjælland, Denmark) at a density of 3 × 10⁴ cells/well in 1% BSA RPMI. If cells were adherent they were left overnight to adhere to wells. All imaging of live cells was performed at the La Trobe Bioimaging Platform on a Zeiss 800 Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany) using 63× oil immersion at 37 °C and 5% CO₂. The duration of imaging is noted in figure legends for respective experiments. Some assays involved the use of fluorescent dyes, including To-Pro-3-APC (Life Technologies), C11-BODIPY 581/591 lipid peroxidation sensor dye (2.5 μM) (Invitrogen, Waltham, MA, USA) and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503) (0.1 mg/mL) (Thermo Fisher Scientific). Images were processed by using software by Zen Image Analysis (Zen Blue Edition version 10.1.19043, Jena, Germany).

25,000 cells of HeLa were seeded per well on an 8-well chamber (Nunc; #155409). To monitor mitochondrial fission, we stained the mitochondria of HeLa cells with 100 nM of Mito-tracker deep red (Invitrogen; M22426) and the nuclei of HeLa cells with 4 nM of Hoechst 33342 (Thermo; #62249) for 30 min. The stained cells were pre-treated with 50 μM of Mdivi-1 (Sigma; M0199) for 1 h, followed by 10 μM of TRIP treatment. HeLa cells were monitored in a humidified atmosphere of 5% CO₂ at 37°C using a high-resolution microscope (Cytiva; Delta Vision).

A549 cells were seeded at a density of 2 × 10⁴ cells/well the day before to allow for adherence in an 8-well chamber (Nunc, Denmark). Cells were stained with 0.05 µg/mL BODIPY and Dye 1 or Dye 2 (250 μM) in 1% BSA RPMI and incubated for 4 h (37 °C and 5% CO₂). After incubation, endpoint images were taken. Imaging was performed on the Zeiss 780 Confocal Laser Scanning Microscope (Zeiss, Germany) at the La Trobe Bioimaging Platform using 63× magnification with oil immersion at 37 °C and 5% CO₂. Spectral imaging-linear unmixing was performed on the Zeiss 780 Confocal Laser Scanning Microscope to separate the fluorescent emission of BODIPY, Dye 1 and Dye 2. Images were analyzed using Zen Image Analysis software (Zen Blue Edition version 10.1.19043).