Nucblue live readyprobes reagent

Cells were plated on 35 mm coverslip bottom dishes (MatTek, Ashland, MA USA). Following a 24 h incubation, cells cultures were dosed at a concentration of 7.5 µg/mL of functionalized Cy5-labeled GNP complex, as well as DTX at a concentration of 2.72 nM. Cell cultures were then incubated for 24 h. NucBlue^™ Live ReadyProbes^™ Reagent (R37605; ThermoFisher Scientific, Waltham, MA, USA) containing Hoechst 33,342 dye and CellLight^™ Tubulin–GFP BacMam 2.0 (ThermoFisher Scientific, Waltham, MA, USA) was used to stain nuclei and microtubules, respectively, prior to imaging. Images were taken using a 60 × oil immersion objective lens using a confocal laser scanning microscope (Zeiss LSM 980, Carl ZeissMicroscopy GmbH, Jena, Germany).

The 3D Matrigel assays were conducted with 1000 cells seeded in Ibidi plates between 2 layers of Matrigel (BD Matrigel Matrix, Growth Factor Reduced (BD Biosciences)) and cultured for 14 days before microscopy analysis (TissueGnostic rig, Vienna, Austria, Europe). Twelve hours post-seeding, 3D embedded cells began to be treated with gels (CARB-F2, CMC-F3, AS-F5) alone and with gels containing 5% (50 µg/mL) CD-NHF (CARB-F4, CMC-F6, AS-F6) for 72 h. After 72 h, the treatments were removed and replaced with normal 3D Matrigel medium (medium corresponding to every cell type supplemented with 2% fetal bovine serum (FBS) and 1% Matrigel). The Live and Dead Cell Assay (Abcam) was used according to the manufacturer’s instructions. Nuclei were counterstained with NucBlue Live Ready Probes Reagent (Thermo Fisher Scientific, Eugene, OR, USA). Fluorescence pictures were acquired at 20× magnification using a Zeiss Axio Observer Z1 Fluorescence Microscope from TissueGnostics rig. Single focal plane images were acquired using Tissue FAXS 4.2 software. The TissueQuest 6.0 software was used for total area segmentation analysis and to quantify the area and sum intensity of fluorescence signal for each spheroid (Figures S4 and S5).

2,000 MEFs expressing mitoYFP were plated in 384‐well and incubated 24 h at 37°C, 5% CO₂. The day of experiment, nuclei were labeled with NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher Scientific) for 30 min at 37°C, 5% CO₂. For stress‐induced fission imaging, cells were treated with 5 μM CCCP or 16 μM 4Br‐A23187 for the indicated time. For stress‐induced hyperfusion imaging, cells were treated with 10 μM CHX or 0.5 μM ActD. Nuclei (NucBlue) and mitochondria (YFP) were imaged every hour for the indicated time using the Operetta CLS High‐Content microscope (PerkinElmer) at 40× Air/0.6 NA. YFP and NucBlue were excited with the 460–490 and 355–385 nm LEDs, respectively. Finally, mitochondrial morphology was quantified as described in the “Mitochondrial morphology quantification” section.