Fig. 2

Non-mitochondrial p66ShcA pools are retained in the cytoplasm and impaired in mitochondrial ROS production. a Wild-type p66ShcA, but not the p66ShcA^S36A mutant, induces ROS production following translocation to the mitochondria. Representative flow cytometry histograms of MitoSOX Red fluorescence in p66-CR (VC), p66-CR (WT), and p66-CR (S36A)-expressing 537-4T1 lung-metastatic breast cancer cells following a 2-h treatment with DMSO (unstimulated) or Actinomycin D. b Geometric mean of MitoSOX intensity for p66-CR (VC), p66-CR (WT), and p66-CR (S36A)-expressing 537-4T1 breast cancer cells treated with Actinomycin D. Each point depicts an independent experiment (n = 6 total). c Schematic depiction of the treatment protocol applied to p66-WT-BirA or p66-S36A-BirA expressing 537-4T1 lung-metastatic breast cancer cells prior to immunofluorescence staining to detect biotinylated proteins. d Quantification of streptavidin immunofluorescence intensity (biotinylated proteins) within mitochondria normalized to streptavidin immunofluorescence in the cytoplasm following treatment with DMSO (unstimulated) or Actinomycin D. e Representative merged images of streptavidin and Tom20 localization in p66-WT-BirA and p66-S36A-BirA expressing 537-4T1 breast cancer cells treated with Actinomycin D. Scale bar is 5 μm. For panel b, statistical analysis was performed using a one-way ANOVA with a Tukey’s multiple comparisons test (*P < 0.05). For panel d, statistical analysis was performed using a two-way ANOVA with a Tukey’s multiple comparisons test (**P < 0.01; ***P < 0.001)