FIG 3.
PB2s with N9 or T9 are imported into mitochondria. (A) HEK 293T cells expressing untagged PB2 with D9 or N9 or APEX-tagged PB2 with D9, N9, or T9 were analyzed by electron microscopy. Scatter plots show the results of densitometry analysis of the mitochondrial and nuclear contrast of individual cells relative to their cytoplasmic contrast. Each point represents a single cell. Data were gated into categories of low and high mitochondrial contrast (y axis) and low and high nuclear contrast (x axis) based on the contrast of mitochondria and nuclei of cells expressing untagged PB2. A diagrammatic key displaying axis titles and values for scatter plots is shown at the top. Staining phenotypes of cells for each quadrant are also shown (white, light mitochondria/light nucleus; blue, light mitochondria/dark nucleus; green, dark mitochondria/light nucleus; gray, dark mitochondria/dark nucleus). N, position of the nucleus; a.u., arbitrary units. (B) Quantitative analysis of cells expressing untagged PB2 with D9 or N9 or APEX-tagged PB2 with D9, N9, or T9, categorized as having either light nuclei (ln) or dark nuclei (dn) and light mitochondria (lm) or dark mitochondria (dm), based on the gating performed as described for panel A. Column data represent percentages of cells expressing the indicated PB2 belonging to the indicated staining categories. Bars represent standard deviations of averages of data from three independent experiments. Asterisks indicate a significant difference between samples (one-sample t test; **, P < 0.01). (C) Comparison of mitochondrial signals of cells with light (ln) or dark (dn) nuclei expressing the indicated PB2, based on the gating performed as described for panel A. Column data represent averages of mitochondrial contrast data from each population. Bars represent the standard deviations of averages of contrast data from three independent experiments. Asterisks indicate a significant difference between samples (one-sample t test; **, P < 0.01; *, P < 0.05; ns, P > 0.05).