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Fig. 1 | BMC Molecular and Cell Biology

Fig. 1

From: Temporal integration of mitochondrial stress signals by the PINK1:Parkin pathway

Fig. 1

Complete and partial depolarization of mitochondria through titration of CCCP. a fluorescent timelapse microscopy images of TMRM-stained HeLa cells at the indicated times post 10 or 5 μM CCCP treatment. b CCCP treatment regimes (top) and corresponding single-cell trajectories of TMRM fluorescence in HeLa cells (bottom). Data from 4 representative cells is shown for each treatment. Trajectories for each cell are represented in a different colour. c population average trajectories of TMRM fluorescence for cells depicted in (b). d average TMRM staining for cells 120 min post-CCCP treatment. Data is presented as an average of 3 biological repeats with a minimum of 18 cells per condition. For (c) and (d), error is represented as the standard error (S.E.). e + f HeLa cells expressing EYFP-Parkin (green) were incubated with the indicated concentrations of CCCP for 24 h, fixed, and stained with DAPI (blue) and antibodies to detect mtDNA nucleoids (red), then imaged by confocal microscopy. Representative images are shown in (e). The average number of mtDNA puncta were quantified for 20 cells per condition and presented in (f) normalized to the number of puncta in control cells. g CCCP treatment regimes (top) and corresponding single-cell trajectories of TMRM fluorescence in SH-SY5Y cells (bottom). Data from 4 representative cells is shown. h fluorescence images of SH-SY5Y cells stained with MitoTraker Green FM (green) and Hoechst 33342 16 h post-CCCP. i, quantification of average mitochondrial mass for cells exposed for 16 h to the indicated concentrations of CCCP. Staining intensity was normalized to the population average for the control cells and presented as the average of the independent repeats. Error is represented as the S.E.. Data is from a minimum of 131 cells per condition across 5 biological repeats. For (d, f, and i) statistical differences in TMRM fluorescence between treatment conditions was appraised by one-way ANOVA and Tukey’s multiple comparisons follow-up test. Statistical significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001. Scale bars represent 50 μm in (a + g) and 20 μm in (e)

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