Fig. 3 |. The predominant endocytic trafficking pathway of AuQC₇₀₅ in MdA-MB-231 human breast cancer cells and tumours is macropinocytosis.

a, The effects of pharmacological inhibitors; n = 3 biologically independent samples. b, Flow cytometry analysis corresponding to a with typical single-parameter gating for approximately 50% of the positive control without inhibitors shows inhibition of internalization. c, Direct and competitive uptake assays of AuQC₇₀₅ in MDA-MB-231 cells demonstrated typical features of macropinocytosis; n = 3 biologically independent samples. While increasing concentrations of AuQC₇₀₅ led to increased uptakes, no obvious blocking effects were observed in the presence of competitive α-LA. d, A three-dimensionally (3D) rendered image of a confocal z stack of MDA-MB-231 cells overlaid with a bright-field image. AuQC₇₀₅ (red) and the macropinocytosis marker FITC–dextran (green) showed a high degree of cytosolic colocalization (yellow) at 2 h. Scale bar, 10 μm. A complete time course for organelle trafficking is shown in Supplementary Fig. 18. e, Ultrastructure analysis with TEM showing an early-formed multivesicular non-homogeneously large-sized (>0.2 μm) and irregularly shaped macropinosome vesicle in MDA-MB-231 cells after 1 h uptake. The cargo is close to the plasma membrane, indicating membrane closure and separation. AuQC₇₀₅ maintained in ultrasmall size can be clearly observed as black dots in the vesicle without apparent aggregation, thereby preserving fluorescence. Scale bars, 2 μm (left), 200 nm (middle) and 50 nm (right). f, Immunofluorescence images of MDA-MB-231 tumours from a J:NU mouse 1 h after injection of AuQC₇₀₅; DAPI was used to visualize nuclei (blue). The organelle trafficking of AuQC₇₀₅ (red) and intracellular early endosome–macropinosome marker EEA1 (green) were in punctate patterns. Colocalizations are indicated by white arrows. Scale bars, 20 μm. g, The majority of AuQC₇₀₅ (red) was found to end up in late endosomes and lysosomes in tumours 2 h after injection, as observed from the extensive colocalization (yellow) with LAMP1 (green). Scale bars, 100 μm.