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. 2021 Apr 12;31(7):1403–1416.e7. doi: 10.1016/j.cub.2021.01.028

Figure 5.

Figure 5

SEVs secreted by PDAC cells with amplified centrosomes activate pancreatic stellate cells

(A) Experimental flowchart.

(B) Representative confocal images of PSCs stained for αSMA (green) and DNA (cyan). Scale bar, 10 μm.

(C) Quantification of the percentage of PSCs activated upon treatment with SEVs collected by UC from PaTu-S.iPLK4 (left) and HPAF-II.iPLK4 (right), with (+DOX) and without (−DOX) extra centrosomes. PaTu-S.iPLK4 isolated SEV: PSCs n(−DOX SEVs) = 398; n(+DOX SEVs) = 373; and n(ctr) = 475. HPAF-II.iPLK4 isolated SEV: PSCs n(−DOX SEVs) = 914; n(+DOX SEVs) = 1,057; and n(ctr) = 718.

(D) Representative confocal image of PSCs incubated with SEVs. Cells were stained for f-actin (phalloidin, gray) and DNA (cyan). Isolated SEVs were labeled with BODIPY (green). Inset depicts higher magnification of sEVs associated with PSCs. Scale bar, 10 μm.

(E) Representative images of cells acquired with the ImageStream. Cells (gray, bright field) and internalized SEV labeled with CellVue (red) are shown.

(F) Quantification of the percentage of PS1 cells positive for CellVue labeling. n(unstained) = 6,280, n(cond. medium) = 6,417, n(−DOX sEVs) = 7,066, and n(+DOX sEVs) = 7,230.

(G) Quantification of the percentage of PSCs activated upon treatment with SEVs collected by UC followed by SEC from PaTu-S.iPLK4 (left) and HPAF-II.iPLK4 (right), with (+DOX) and without (−DOX) extra centrosomes. PaTu-S.iPLK4 isolated SEV: PSCs n(−DOX SEVs SEC7) = 161; n(+DOX SEVs SEC7) = 154; PSCs n(−DOX SEVs SEC8) = 490; n(+DOX SEVs SEC8) = 387; PSCs n(−DOX SEVs SEC9) = 463; and n(+DOX SEVs SEC7) = 454. HPAF-II.iPLK4 isolated SEV: PSCs n(−DOX SEVs SEC7) = 499; n(+DOX SEVs SEC7) = 410; PSCs n(−DOX SEVs SEC8) = 541; n(+DOX SEVs SEC8) = 713; PSCs n(−DOX SEVs SEC9) = 1,035; and n(+DOX SEVs SEC7) = 914.

For all graphics, error bars represent mean ± SD from three independent experiments. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and n.s. (p > 0.05). For all, graphs were analyzed using two-way ANOVA with Tukey’s post hoc test. See also Figure S5.