Figure 2. Breast cancer-derived sEV^HYP regulate mitochondrial dynamics.

(A and B) MCF10A cells treated with T47D cell-derived sEV^NORM or sEV^HYP were imaged after 24 h for subcellular mitochondrial distribution by confocal fluorescence microscopy (A, representative images) and the percentage of mitochondria localized to the cortical cytoskeleton was quantified (B). Insets, magnification of indicated areas. Scale bar, 5 μm. Mean±SD (N=3). Numbers correspond to p values by 1-way Anova with Tukey’s posttest.

(C) MCF10A cells treated with MCF7- or T47D-cell-derived sEV^NORM or sEV^HYP were analyzed for changes in mitochondrial inner membrane potential by TMRM labeling and flow cytometry. Mean±SD (N=3).

(D) MCF10A cells treated with MCF7 cell-derived sEV^NORM or sEV^HYP for 8 h were analyzed by Western blotting. p, phosphorylated. (N=3).

(E and F) sEV^NORM- or sEV^HYP-treated MCF10A cells were analyzed for co-localization of Ser616-phosphorylated Drp1 (pDrp1) and mitochondria by confocal microscopy (E, representative images) and quantified (F). Insets, image magnification of indicated areas. Mean±SD (N=3). Scale bar, 5 μm. Numbers correspond to p values by 1-way Anova with Tukey’s posttest. PCC, Pearson Correlation Coefficient.

(G and H) sEV-treated MCF10A cells were analyzed for changes in mitochondrial volume indicative of organelle fusion (>1.5-fold, positive y-scale) or fission (<1.5-fold, negative y-scale) over 30-sec intervals (G) and the number of mitochondrial fusion and fission events was quantified (H) Each line corresponds to changes in mitochondrial volume in a single cell. Mean±SEM (N=3). Numbers correspond to p values by 2 way-Anova with Sidak's multiple comparisons test. See also Figure S3.

(I and J) sEV-treated MCF10A cells transfected with control non-targeting siRNA (siCtrl) or siRNA directed to MNF1 (siMFN1) or Drp1 (siDrp1) were analyzed by Western blotting (I) and cell migration on PET inserts (J). Mean±SD (N=3).