In carta image analysis software

To assess cell membrane integrity, as a marker of cell death, PI fluorescent staining was performed using PI dye. To stain MB spheres, 100 μL of media was removed from each well and PI at a concentration of 40 μg/mL in PBS (100 μL) was added to the treated spheres and analysed under a fluorescent microscope (IN Cell Analyzer 2000, GE Healthcare, IL, USA). ImageJ 1.49q software (NIH, Bethesda, MD, USA, website: https://imagej.nih.gov/ij/) was used to analyse PI incorporation by measuring the signal intensity of the sphere from the images. For live/dead dual staining, treated spheres were dissociated into single-cell suspension using Accutase solution (A6964, Sigma-Aldrich). Subsequently, cells were washed with PBS and stained with 3 μM calcein-AM (Thermo Fisher Scientific) and propidium iodide (5 μM) followed by incubation for 30 min. One hundred microlitres of cell suspension was transferred to black plates and imaged with fluorescent microscope (IN Cell Analyzer 2000) using excitation of 475 nm/emission of 511 nm for calcein-AM and excitation of 542 nm/emission of 620 nm for PI. Generated images were automatically analysed using IN Carta Image Analysis Software (GE Healthcare).

To assess DNA damage, 5 × 10³ cells were seeded in 100 µL serum-free DMEM per well in 384-well plates (781091, µClear, Greiner, Ryde, NSW, Australia) pre-coated with rat tail collagen as described above and left to adhere overnight. The cells were treated with test compounds for 4 h (0–40 μM in 100 μL HBSS/well) while menadione was used as a positive control [32 (link)]. After fixation (50 μL) and permeabilization (50 μL) as described above, unspecific antibody binding was blocked for 1 h (5% FBS + 5% BSA in PBS, 50 μL/well) before the samples were exposed to mouse monoclonal anti-phospho-Histone H₂AX (Ser139) antibody overnight (1:1000 in blocking buffer, 15 μL/well). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody for 1 h (1:10,000 in PBST, 15 μL/well), nuclei were counterstained using DAPI and stored for imaging and analysis as described above. The average numbers of γ-H₂AX-positive cells were automatically quantified for all acquired images using IN Carta image analysis software (GE Healthcare, Rydalmere, NSW, Australia). Results were standardized on the non-treated control and expressed as fold-change. Data represented the mean ± SEM of quadruplicate images from 3 independent assays. At least 1000 cells were analysed per treatment.

To measure cellular Hsp70 protein levels, cells were seeded, treated, fixed, and permeabilized as described under 4.6. Celastrol [83 (link)] and shikonin [84 (link)] were used as positive control compounds. After blocking, cells were exposed to rabbit monoclonal anti-Hsp70 antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-rabbit Alexa Fluor 594 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular Hsp70 intensity was automatically quantified for each acquired image. Data were standardized on the non-treated control (100%) and expressed as mean ± SD of at least quadruplicates from one assay. At least 1 × 10³ cells were analyzed for each treatment.

Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzim-idazolylcarbocyanine iodide (JC-1) Mitochondrial Membrane Potential Assay Kit (ab113850, Abcam) according to the manufacturer’s instructions. Briefly, MB cells were washed twice and incubated with the assay solution containing JC-1 dye (10 μM) for 30 min. Cells were then washed, suspended in assay buffer (supplemented with 5% FBS) and seeded at 6 × 10⁴ in 50 μL in a black 96-well plate (655,090, Greiner, Germany). Fifty microlitres of 2 × concentration of treatments, including different concentrations of mibefradil, NNC and carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) as a positive control, or no treatment control, was added to cells followed by incubation at 37 °C for 6 h. Cells were imaged using IN Cell Analyzer 2000 (excitation wavelength used was 475 nm and emission wavelengths were 511 nm and 587 nm for the monomer and the aggregates of JC-1 molecules, respectively). Images were analysed using IN Carta Image Analysis Software (GE Healthcare).

To assess if the test compounds induce some level of genotoxicity, cells were seeded as previously described and treated with 10 μM test compounds. Since γ-H2AX signal can decrease over time due to DNA repair, this assay captured SCQ-induced DNA damage after a short treatment period of 4 h. Celastrol was used as a positive control [87 (link)]. After fixation, permeabilization and blocking, cells were exposed to mouse monoclonal anti-phospho-Histone H₂AX antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). The number of γ-H₂AX-positive cells was automatically quantified for all acquired images. Percentage γ-H₂AX-positive cells was expressed as mean ± SD of at least quadruplicates from one assay. At least 500 cells were analyzed for each treatment.