Fig 1: Quantitative assessment of Bcl-2 family protein localization to the mitochondria in live cells. (a and b) HEK293T cells were transfected with fluorescent protein fusions to Bcl-2 family members, stained with MitoTracker dye (blue) and visualized live using confocal microscopy. (a) Localization of pro-survival Venus-Bcl-2 family constructs (green). The mitochondrial targeting peptide of subunit VIII of cytochrome c oxidase fused to the carboxy-terminus of Venus (Venus-mito) was used as a positive control and Venus alone was used as a negative control. (b) Localization of mCherry- or Venus- (in the case of Noxa) tagged-BH3-only proteins (red). (c) Two different exposure images exemplifying the differential intensities of MitoTracker staining in the mitochondria and cytoplasm that were used as the basis for image analysis depicted in d. (d) Image analysis protocol used to quantify mitochondrial targeting of fluorescent fusion proteins. MitoTracker staining (blue in raw data) was used to mask the cytoplasm, nucleus and mitochondria (red, blue and green, respectively, in Mito masking). Cells within an appropriate range of expression levels for quantification were identified (transfection mask) and fluorescence intensities were measured in these cells (cells analyzed). (e) Measurement of mitochondrial targeting for each pro-survival (green) and pro-death (red) Bcl-2 super-family fluorescent construct. Mitochondrial targeting is denoted by the ratio of mitochondrial to cytoplasmic fluorescence intensity measured according to the protocol described in d. A ratio of one represents no detectable mitochondrial targeting. Scale bars 10 µm