The mGold, a yellow fluorescent protein, was clone from pCMV-mGold-Actin-C-18 (MiaoLingBio; P50209) (Supplementary Fig. 16). The COX8 and Halo tag was synthesized (by Tsingke Biotech, China) and then fused with mGold by referencing mEmerald-Mito-7 (plasmid 54160, Addgene) using seamless cloning. For three-color mGold-HaloTag-Lysosome tracking, cells were further incubated with 50 nM LysoTracker™ Deep Red (L12492, Thermo Fisher Scientific) working solution for 20 min at 37 °C to enable precise visualization. The subsequent co-localization imaging (Supplementary Figs. 27 and 28) or tracking of mGold, JF549, and LysoTracker Deep Red was performed via confocal fluorescence microscopy (LSM 980, Zeiss; Dragonfly CR-DFLY-202-40, and Dragonfly CR-DFLY-202-2540, Nikon) or 3D-SpecDIM.
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