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Fig. 2 | Molecular Cancer

Fig. 2

From: Intercellular transfer of exosomal wild type EGFR triggers osimertinib resistance in non-small cell lung cancer

Fig. 2

Exosomal wtEGFR protein can be internalized by sensitive mutEGFR cancer cells via Clathrin-mediated pathway. a Western blot analysis of indicated exosomal markers (positive markers: CD63, TSG101, Alix; negative marker: Calnexin) in H1299 whole cell lysates and H1299 cells-derived exosomes. b Nanoparticle Tracking Analysis of H1299 cells-derived exosomes. c Transmission Electron Microscope imaging of H1299 cells-derived exosomes. Scale bar: 100 nm. d Western blot analysis of colocalization of exosomal marker CD63 and transmembrane protein EGFR in various extracellular vesicle fractions as separated by sucrose density gradient centrifugation. e Western blot analysis of EGFR protein level of H1975 cells after pre-incubation with CM of H1299 cells, CM dExo of H1299 cells and H1299 cells-derived exosomes. f Real-time quantitative analysis of EGFR mRNA level of H1975 cells after pre-incubation with CM of H1299 cells, CM dExo of H1299 cells and H1299 cells-derived exosomes. g Representative images of internalization of H1299 cells-derived exosomes containing GFP-tagged wtEGFR in H1975 cells (RFP). Scale bar: 20 μm. (H) Western blot analysis of Caveolin-1 protein expression to verify the efficacy of Caveolin-1 silencing by shRNAs. i Western blot analysis of Clathrin protein expression to verify the efficacy of Clathrin silencing by shRNAs. j-k Flow cytometric exosomes absorption assay of control PC9 cells or Caveolin-1- and Clathrin-silenced counterparts treated with PKH-67-labeled exosomes (5 μg/mL) for 12 h. l-m Flow cytometric exosomes absorption assay of PC9 cells pre-cultured with various concentration of CPZ for 2 h following PKH-67-labeled exosomes (5 μg/mL) incubation for another 12 h. All data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

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