Extended Data Fig. 4: TEVs-mediated intercellular communications traced by NP1.
a, CLSM images of MDA-MB-231 cells after incubation with NP1 for 1 h in the absent (untreated) of FP. Scale bar, 10 μm. b, Cell viabilities of MDA-MB-231 cells after treatment with PBS (untreated control, Unt), wortmannin, cytochalasin D, sucrose, dynasore, and lipid raft inhibitors (MβCD and Nystatin) at 37°C, as well as PBS at 4 °C, respectively. c, Representative confocal fluorescence images for observing the colocalization of NP1 (red) with lipid raft marker CTxB (green). MDA-MB-231 cells were treated with NP1 for 1 h. Scale bar, 10 μm. d,e, Experimental schemes of transwell systems for co-culture of donor tumour cells (cells incubated with NP1 (d) or Ctrl-NP1 (e)) and recipient tumour cells. f, CLSM images of the uptake of TEVsNPs (TEVsNP1 or TEVsCtrl-NP1) by recipient tumour cells after co-incubation with donor tumour cells for 12 h. DAPI is used to stain nuclei. NP1 and Ctrl-NP1 are shown in red. Scale bars, 10 μm. g, Representative CLSM images of NIH-3T3 cells pretreated with NP1 for 1 h co-cultured with NIH-3T3EGFP cells for 4 h. Scale bar, 20 μm. The results showed that after 4 h of co-culture, both the fluorescence intensity of the donor cells (NIH-3T3 cells) and recipient cells (NIH-3T3EGFP cells) were very weak but observable, reflecting the inefficient cellular uptake by normal cells and inter-normal cell communications. h, Effect of GW4869 (one inhibitor of SEVs secretion, 10 μM) on the intercellular communication between MDA-MB-231 cells and MDA-MB-231EGFP cells observed by confocal microscopy. Scale bar, 5 μm. i, Statistical analysis of the relative MFI of MDA-MB-231EGFP cells after different treatments. In all experiments, the concentration of NP1 and Ctrl-NP1 was 0.1 g/L. In b, n = 4 biologically independent samples; in i, n = 3 biologically independent samples. Data are presented as the mean ± s.d. Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test (b) and unpaired two-tailed Student’s t-test (i). In a-c,f-i, experiments were repeated three times independently with similar results; representative images are shown in a,c,f,g,h.
