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. 2016 Feb 1;27(3):466–482. doi: 10.1091/mbc.E15-09-0632

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© 2016 Mills et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Loss of APC reduces initiation of mitochondrial transport but not velocity in NIH 3T3 cells. Cells were treated with either control or red fluorescently tagged mouse APC siRNA (mAPC-red), transfected with a mitochondrial marker (pGFP2-Mito), and then grown to confluence. Cells were then wounded and were analyzed 5 h later by live-cell time lapse, with images captured every 7 s for a total of 5 min. Post image acquisition, mitochondria were selected at random for tracking manually using the ImageJ plug-in MTrack. (A) Representative cell images are shown from ∼1 min after commencement of tracking. Mitochondria are marked for different types of movement: stationary/oscillating (yellow), anterograde/membrane-directed (green), and retrograde/nucleus-directed (red). The majority of directed movement of mitochondria was blocked when APC was silenced (successful APC silencing indicated by red fluorescence). Scale bars: 10 μm. (B) Confirmation of APC knockdown in fixed cells treated with mAPC-red siRNA (C) Analysis of mitochondrial motility revealed that loss of APC resulted in decreased overall mitochondrial transport (**, p < 0.01). (D) This was particularly evident in the anterograde direction (*, p < 0.05; n.s., not significant). (E) Analysis of mitochondrial velocity (μm/min) revealed that loss of APC had no impact on the speed of movement in either direction once transport was initiated (n.s., not significant). n over three independent experiments: control cells, n = 18, and mitochondria, n = 202; mAPC-red cells, n = 18, and mitochondria, n = 189. Bar graph data presented as mean (±SD), statistical analysis by two-tailed t test (C–E).