Gold fiducials

HeLa cells (cell line 25) were maintained in a humidified 37 °C incubator with 5% CO₂, then cultured in DMEM medium with no phenol red (Gibco), containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For cryo-CLEM and cryo-ET, cells were plated onto fibronectin-coated 200-mesh gold R2/2 London finder Quantifoil grids (Quantifoil Micro Tools) at a density of 2 × 10⁵ cells/mL. After a 12-h incubation, cultures were treated with MG-132 (1 µg/mL) for 2 to 3 h or left untreated, before being plunge-frozen in liquid ethane/propane mixture using an FEI Vitrobot Mark IV (21 (link)). Immediately before plunge-freezing, 3 µL of a suspension of beads was applied to grids. The bead suspension was made by diluting 500 nm blue (345/435 nm) polystyrene fluorospheres (Phosphorex) with a colloidal solution of 20 nm gold fiducials (Sigma-Aldrich) pretreated with BSA. The gold served as fiducial markers for cryo-tomogram reconstruction, and the blue fluorospheres served as landmarks for registering fluorescence light microscopy (FLM) images from different channels as well as cryo-EM images (24 (link)). Plunge-frozen grids were subsequently loaded into FEI Polara EM cartridges. EM cartridges containing frozen grids were stored in liquid nitrogen and maintained at ≤−150 °C throughout the experiment, including cryo-FLM imaging, cryo-EM imaging, storage, and transfer.