Fig. 3: Photoinitiated GA DPRs impaired nucleocytoplasmic transport in human neuroblastoma cells.

a Immunofluorescence images of Ran protein (red) in ADP-2 photoinitiated (+ADP-2 +UV) or control SH-SY5Y cells. GA DPRs aggregates were shown in green color. Cells were treated with ADP-2 (1 μM) and then exposed to UV light (wavelength: 335–379 nm, power density: ≤8.24 mW/cm², duration: 1 min) and incubated for 24 h. Yellow arrows indicate the cells with nuclear Ran depletion. White dash line indicates the region for fluorescence intensity profiling. Scale bars indicate 10 µm. b Fluorescence intensity profile of selected cells in Fig. 3A. Blue curve indicates DAPI channel and red curve indicates Ran channel. c Quantification analysis of SH-SY5Y cells with nuclear ran protein depletion. Three biological replicates were carried out (r = 3). More than 55 cells were counted and analyzed in each group (n ≥ 55). Mean and standard deviation for +ADP-1 +UV = 69.0 ± 9.6; +ADP-1 −UV = 16.2 ± 5.5; −ADP-1 +UV = 14.7 ± 6.0; −ADP-1 −UV = 18.0 ± 4.4. ** indicates statistical significance where p value < 0.01 (analyzed by two-sided Welch’s T test with Bonferroni correction, test statistic in group comparison between +ADP-1 +UV and −ADP-1 −UV: p value = 0.0011, degree of freedom = 3, t value = 3.18; group comparisons between +ADP-1 −UV and −ADP-1 –UV: p value = 0.23, degree of freedom = 4, t value = 2.77; group comparison between −ADP-1 +UV and −ADP-1 –UV: p value = 0.16, degree of freedom = 4, t value = 2.77), and n.s. indicates not significant. d Immunofluorescence images of importin-β (red) in SH-SY5Y after ADP-2 (1 μM) and irradiation treatment. GA DPRs aggregates were shown in green color. Yellow arrows indicate the cells with importin-β mislocalization. White dash line indicates the region for fluorescence intensity profiling. Scale bars indicate 10 µm. e Fluorescence intensity profile of selected cells in Fig. 3D. Blue curve indicates DAPI channel and red curve indicate the importin-β channel. f Quantification analysis of SH-SY5Y cells with importin-β diffusion. Three biological replicates were carried out (r = 3). More than 37 cells were counted and analyzed in each group (n ≥ 37). Mean and standard deviation for +ADP-1 +UV = 62.0 ± 7.0; +ADP-1 −UV = 21.7 ± 6.9; −ADP-1 +UV = 24.1 ± 7.6; −ADP-1 −UV = 17.8 ± 5.8. *** indicates statistical significance where p value < 0.001 (analyzed by two-sided Welch’s T test with Bonferroni correction, test statistic in group comparison between +ADP-1 +UV and −ADP-1 −UV: p value = 0.0004, degree of freedom = 4, t value = 2.78; group comparison between +ADP-1 −UV and −ADP-1 –UV: p value = 0.17, degree of freedom = 4, t value = 2.77; group comparison between −ADP-1 +UV and −ADP-1 –UV: p value = 0.10, degree of freedom = 4, t value = 2.77), and n.s. indicates not significant. g Immunofluorescence images of sGFP-transfected SH-SY5Y cells after ADP-1 treatment (1 μM) and UV-irradiation. Corresponding nuclear importation (Importazole, IPZ, 40 µM) and exportation (leptomycin B, LMB, 20 nM) inhibitors were used here as reference groups, respectively. Scale bars indicate 10 µm. h Quantification analysis of the nuclear-to-cytoplasmic ratio of sGFP reporter in SH-SY5Y. One biological replicate were carried out (r = 1). Cell counting number (n) in each group: control = 38, LMB = 36, IPZ = 31, ADP-1 = 38. Mean and standard deviation for control = 0.98 ± 0.10; LMB = 2.18 ± 0.22; IPZ = 0.54 ± 0.14; ADP-1 = 0.40 ± 0.06. *** indicates statistical significance where p value < 0.001, (analyzed by two-sided Welch’s T test with Bonferroni correction, test statistic in group comparison between control and LMB: p value = 7.6 × 10⁻³³, degree of freedom = 48, t value = 2.01; group comparison between control and IPZ: p value = 3 × 10⁻²¹, degree of freedom = 54, t value = 2.00; group comparison between control and ADP-1: p value = 2.6 × 10⁻³⁸, degree of freedom = 59, t value = 2.00).