Fig. 1: Blebbisomes form with functional mitochondria.
From: Blebbisomes are large, organelle-rich extracellular vesicles with cell-like properties
a, Representative blebbisomes imaged with DIC microscopy. The arrowheads show blebs. b, The diameter of blebbisomes. Average blebbisome diameter: 14.9 ± 3.0 s.e.m., 8.92 ± 0.8 s.e.m., 10.9 ± 1.2 s.e.m. in B16-F1, DKO-1 and MDA-MB-231 cells, respectively. n = 167, 183 and 236 blebbisomes for B16-F1, DKO-1 and MDA-MB-231 cells, respectively. c, An SEM micrograph of B16-F1 blebbisome displaying large characteristic bleb (arrowhead). d, Correlative light and electron microscopy of a B16-F1 blebbisome using iSIM (top and middle) and SEM (bottom). The colour bar denotes the relative Z height, and the dotted line denotes the height of the single z-slice. The arrowheads show the blebs. e, A timelapse DIC and epifluorescence (MitoTracker) microscopy time montage showing blebbisome formation. The arrow denotes the blebbisome formation, and the arrowhead denotes mitochondria. f, Imaging of actin filaments and mitochondria in blebbisomes by iSIM. The boxes indicate blebbisomes. Bottom: enlarged boxes are shown below (n = 3). g, TMRE fluorescence before and after FCCP treatment. The arrow shows a cell, and the arrowhead shows a blebbisome (n = 5). h, A timelapse of TMRE fluorescence before and after FCCP treatment. The fluorescence levels between the cells and blebbisomes after FCCP treatment were not significantly different as determined by a two-tailed Student’s t-test. For DKO-1 post FCCP treatment, blebbisome mean: 0.42 ± 0.46 s.e.m., cell mean: 0.35 ± 0.46 s.e.m. For B16-F1 post FCCP treatment, blebbisome mean: 0.55 ± 0.058 s.e.m, cell mean: 0.47 ± 0.058 s.e.m (n = 5 cells and blebbisomes each for both DKO-1 and B16-F1 cells). i, Kaplan–Meier survival curves for blebbisomes treated with DMSO, FCCP, staurosporine or raptinal. Time of death was denoted by a loss of membrane integrity (Supplementary Video 7). The mean survival time for DMSO, FCCP, staurosporine and raptinal are 23.18 ± 1.23 s.e.m., 19.59 ± 1.57 s.e.m., 20.36 ± 1.46 s.e.m. and 20.46 ± 1.68 s.e.m., respectively. DMSO was significantly different from FCCP, staurosporine and raptinal with a P value of 2.3 × 10−5, 0.00041 and 0.00040, respectively, as determined by a two-tailed Student’s t-test (n = 29 for DMSO, 41 for FCCP, 29 for staurosporine and 39 for raptinal; across three independent experiments).
