Figure 1.
Schematic of digitonin permeabilization of plasma membrane to detect redistribution of cyt c and Smac by flow cytometry.
Cells are permeabilized with digitonin followed by paraformaldehyde (PFA) fixation. Primary antibodies specific for target proteins cyt c or Smac, and fluorescently-tagged secondary antibodies, are applied to the cells followed by washing steps to remove unbound antibodies. (Left side) Flow cytometry detects cells that retain cyt c (red) or Smac (green) in mitochondria through the binding of fluorescently-tagged antibodies. (Right side) Cells that have redistributed either cyt c or Smac to the cytosol lose almost all of each protein through the permeabilized plasma membrane (PM) (dotted line).
Figure 2.
Confocal microscopic imaging of single cells.
All images shown are from cultures of 143B cells treated with STS for 24 h. (A) Representative images of cells permeabilized with digitonin, showing retention or redistribution of cyt c or Smac from mitochondria. Cells were processed as for flow cytometry, then immunostained with antibodies specific for cyt c and Smac. Cells were then stained with DAPI (to visualize the nucleus) and finally centrifuged on coverslips prior to confocal microscopy. Images were acquired by confocal microscopy using a FluoView 500 with 60x objective lens. Overlay 1 is a merge between DAPI stain (blue channel) and cyt c (red channel); Overlay 2 is a merge between DAPI stain (blue channel) and Smac (green channel). (B) Representative images of 143B cells subjected to direct immunocytochemical procedures with antibodies specific for cyt c and Smac, and also DAPI-stained, showing retention or redistribution of cyt c or Smac. Cells were grown in 6-well plates, then centrifuged to recover both detached and adherent cells, which were then fixed with 3.5% paraformaldehyde and treated with Triton X-100 prior to immunostaining. Slightly different nomenclature is used to describe the complementary attributes of the top rows of cells in (A) and (B) because flow cytometry detects cells that retain the target protein while direct immunocytochemistry scoring is based on the observed redistribution of target proteins from mitochondria to the cytosol (but still within the boundary plasma membrane). Arrow indicates relevant cell in Row B2. Scale bar (20 µm) applies to all images in each of panels A and B. Very occasionally, significant amounts of Smac may appear in nucleus (cf. ref [29]), but this is extremely rare, with less than 3% of cells showing such localization of Smac (data not shown).
Figure 3.
Dot plot profiles of untreated and STS-treated cells.
(A) Schematic dot plot diagram showing the key to quadrant division. Each quadrant represents the apportionment of cells that retain both cyt c and Smac in mitochondria or have redistributed either or both of these proteins. (B) The populations of cells for each of the staining regimes, with the specified application of antibodies (Ab), are gated in the forward scatter (FSC) and side scatter (SSC) dot plots to eliminate dead cells and cell debris, using the “No staining” population (no antibodies applied) as the basis for this R1 gate. (C) Fluorescence dot plot analysis within the R1 gate. Populations corresponding to “No staining” (autofluorescence) and “Secondary Ab only” (non-specific binding), are located in the lower left quadrant. Cells stained with both primary and secondary antibodies are overwhelmingly located in the top right quadrant (these cells were not exposed to apoptotic inducer). Quadrant delineation is described in the text. (D) Gating of cells (R3) treated with STS for 24 h; all other indications as for panel B. Note that the R2 gate in this experiment corresponds to the 6-h time point for STS treatment, data for which are not shown here. (E) Dot plot analyses for cells treated with STS for 24 h. All indications correspond to those of panel C. Red and purple colors in panels C and E, respectively, are generated by CellQuest software (i.e. neither color refers to cyt c, specifically).
Figure 4.
Differential redistribution of cyt c and Smac in 143B cells treated with STS.
143B cells were untreated or treated with STS (100 nM) for various times. (A) Flow cytometry data corresponding to the quantified quadrant occupancy in Figure 3C and F, averaged over three independent experiments: (i) indicates individual cell scoring for cells in each of four categories; (ii) indicates cumulative scoring for each of cyt c and Smac. Data for 3,000 events were collected for flow cytometry analysis in each individual experiment at each time point. (B) Direct immunocytochemical analyses of cells under four categories as indicated for each time point. 300 cells were scored for immunocytochemical analysis for each time point in each individual experiment. (i) and (ii) are as above for Panel A. Each standard error bar represents ± SEM from three independent experiments. Asterisks indicate significant differences of cyt c and Smac redistribution (*P<0.05; **P<0.005; ***P<0.0005).
Figure 5.
Simultaneous redistribution of cyt c and Smac in HeLa cells treated with STS.
HeLa cells were untreated or treated with STS (300 nM) for various times. All other indications as for Figure 4.
Figure 6.
Early redistribution of Smac relative to cyt c in 143B cells treated with MT-21.
143B cells were untreated or treated with MT-21 (200 µM) for various times. All other indications as for Figure 4.