The non-classical nuclear import carrier Transportin 1 modulates circadian rhythms through its effect on PER1 nuclear localization
Fig 6
Nuclear localization is of PER1 is modulated by oxidative stress in a TNPO1-dependent manner.
(A) Steady-state subcellular localization of ectopically expressed PER1- or PER2-Venus fusion proteins in TNPO1 depleted (red) or ns control (black) U-2 OS cells with or without H2O2 treatment. Depicted are nuclear to cytoplasmic fluorescence intensity ratios determined in four individually performed experiments (box: median ± 25 percentile; whiskers: 10–90 percentile, n = 185 to 801, One-way-ANOVA, posttest: Tukey’s multiple comparison test: n.s. = non-significant, *** p < 0.001). (B) Steady-state subcellular localization of ectopically expressed truncated versions (see also Fig 3D top) of PER1-Venus fusion proteins in U-2 OS cells with or without H2O2 treatment. Depicted are nuclear to cytoplasmic fluorescence intensity ratios (box: median ± 25 percentile; whiskers: 10–90 percentile, n = 21 to 59 cells, One-way-ANOVA, posttest: Tukey’s multiple comparison test: n.s. = non-significant, *** p < 0.001). (C) Kinetics of nuclear import upon H2O2 treatment was analyzed using fluorescence recovery after photobleaching (FRAP) of individual nuclei. Upper part: representative images with arrow heads indicating bleached nuclei (scale bar = 20 μm). Lower part, left: For quantification nuclear fluorescence intensity (normalized to cytoplasmic intensity) was determined per time point (left panels). Given are means ± SEM, n = 15 to 18 individual cells. For comparison, data for non-silencing control are re-plotted from Fig 4C. Two-way-ANOVA revealed indicated significance between treatment groups (*** p < 0.001, n.s. = non-significant). Lower part, right: The average recovery time of 25% of initial fluorescence intensity was calculated. Given are means of the times for 25% of initial PER1-Venus fluorescence recovery after photobleaching nuclei from Tnpo1depleted (red) or ns control (black) cells (error bars = SD, n = 10–17 cells for each condition, one-way ANOVA with Bonferroni-Holm corrected posthoc Student’s t-test (one-sided); * p < 0.05; n.s. = non-significant). Data of the two leftmost columns are re-plotted from Fig 4C.