Fig 2: Bivariate histograms of LB1 fiber–NPC and face center–NPC distances. (A) First row shows a bivariate distribution of NPC to LB1 fiber and face center distances in WT MEFs after treatment with scramble siRNA. (B) Second row shows the same with siRNA KD of TPR. (C) Third row shows the same with siRNA KD of NUP153. (D) Fourth row shows the same with siRNA KD of ELYS. (A–D) First column represents the observed bivariate distribution. Second column represents the expected bivariate distribution. Third column represents the difference between expected and observed. Difference between the observed and expected distance distributions with purple indicating where the observed exceeds the expected frequency (Freq) and green showing when the observed frequency is less than the expected frequency. Marginal violin plots and box plots of the distances correspond with the half-violin plot counterparts of the same orientation and color as in panels B–E of Fig. 7. Each violin or box plot represents 20 cells with the number of NPCs detailed in Table 3.

Fig 3: Phosphoproteomics revealed decreased phosphorylation of Nup153 in Tpr knockdown cells. (A) The workflow of phosphoproteomics. (B) Label-free quantification of all of the phosphopeptides obtained (5282 phosphopeptide species) was performed. The intensity based on the peak areas of phosphopeptides was calculated with Skyline pipeline. The intensity in the control knockdown condition was divided by the intensity of the same phosphopeptide in the Tpr knockdown condition to obtain differential phosphorylation levels between two conditions (i.e., decreased phosphorylation in the Tpr knockdown condition). Phosphopeptides were ranked according to log2 ratio. The Y-axis indicates log2 value. (C) Label-free quantification of two different phosphopeptides derived from Nup153 was performed by Skyline. The Y-axis indicates total signal intensity. (D) The amount of total peptides that represents the protein level was analyzed using Skyline pipeline. All of the peptides derived from either Nup153 or Tpr before phosphopeptide enrichment were quantified and summarized. The total abundance of Nup153 peptides did not change in both knockdown conditions, while Tpr peptides were significantly reduced in Tpr knockdown condition.

Fig 4: Prolonged loss of TPR during senescence blocks NF-κB activation.(A) TPR and NF-κB immunostaining in control (STOP) and oncogene-induced senescence (OIS) (RAS) cells after 4-hydroxytamoxifen (4-OHT) and siRNA (control and TPR) treatment for 8 days. Scale bar: 10 μm. (B) Quantification of NF-κB nucleocytoplasmic ratios in experiment described in (A). Kruskal-Wallis testing was used to determine statistical significance followed by Dunn’s post hoc testing. n.s. p>0.05, ***<0.001. (n) indicates the number of cells analysed for each sample. Data from a biological replicate are in Figure 2—figure supplement 1A. Statistical data are in Figure 2—source data 1. (C) Immunoblots of extracts from control (STOP) and OIS (RAS) cells after 4-OHT and siRNA treatment for 8 days for phosphorylated (pS536) and total NF-κB with vinculin as a loading control. Numbers below indicate the ratio of band intensity for NF-κBpS536 or NF-κB and the vinculin loading control with the ratio for RAS siCTRL normalised to 1.00. (D) As in (C) but for phosphorylated (pS176/180) IKKα/β and total IKKα and with β-actin as a loading control. Data from biological replicates of (C) and (D) are in Figure 2—figure supplement 1B and C. (E) Above: Schematic of controlled media experiment to investigate whether TPR loss causes a general defect in NF-κB transport. STOP and RAS cells were grown for 8 days and treated with 4-OHT and siRNAs. On day 8 (d8) they were treated for 45 min with conditioned media (CM) taken from either STOP or RAS cells grown in 4-OHT-containing media for 8 days. Below left: NF-κB immunostaining in STOP or RAS cells treated with CM harvested from STOP (left) or RAS (right) cells. Scale bar: 50 μm. Below right: Same experiment with images shown at greater magnification. Scale bar: 10 μm. (F) Quantification of NF-κB nucleocytoplasmic ratios for experiment shown in (E). Data from a biological replicate are in Figure 2—figure supplement 1D. Statistical data are in Figure 2—source data 1. Figure 2—source data 1.Quantification of NF-κB nucleocytoplasmic ratios and statistical analysis for data in Figure 2B and F, and for biological replicates in Figure 2—figure supplement 1A and D.Median NF-κB nucleocytoplasmic ratios (n/c) and number of cells analysed for day 8 (d8) STOP or RAS cells subject to knockdown with control (CTRL) or TPR siRNAs, and for experiments where these cells were treated with conditioned media (CM) from either STOP or RAS cells. Kruskal-Wallis testing was used to determine statistical significance for each replicate (p-value in parentheses) followed by Dunn’s post hoc testing. p-values after Benjamini and Hochberg correction. Figure 2—source data 2.Uncropped and labelled gels for Figure 2. Figure 2—source data 3.Raw unedited gels for Figure 2.

Fig 5: Translocated promoter region (Tpr) phosphorylation determines subcellular localization in neural stem/precursor cells (NSPCs). (A) Scheme illustrating the localization of Tpr and phenylalanine-glycine repeat nucleoporins (FG-Nups) in nuclear pore complexes (NPCs). (B) Comparative imaging of NPCs in subventricular zone- (SVZ-) derived NSPCs immunolabeled with Tpr (red) and FG-Nups (green) using confocal laser scanning microscopy (CLSM) (left) and SR Airyscan microscopy (right). Enlargements at the right of each panel indicate representative resolutions of the Tpr and FG-Nup signals, and white arrowheads indicate the distinct localizations of TPR and FG-Nups resolved with SR Airyscan microscopy. Nuclei are stained with DAPI (blue). Scale bars: 2 µm, 500 nm (enlargement). Representative images from three independent experiments (10 nuclei were analyzed in total). (C) Scheme illustrating Tpr localization relative to FG-Nups in an NPC after SR Airyscan microscopy and Imaris 3-D processing. (D) Representative SR Airyscan microscopy and Imaris-processed image of an SVZ-derived NSPC nucleus immunolabeled for Tpr (red) and FG-Nups (green). Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 2 µm. (E) 3-D visualization revealing distinct localizations for Tpr (red) and FG-Nups (green) in the NPC of SVZ-derived NSPCs. Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 1 µm. (F) 3-D visualization of confocal images revealing nuclear phospho-Tpr (P-Tpr, red) localization in NSPCs in vitro. Representative images from three independent experiments (10 nuclei were analyzed in total). Scale bars: 2 µm, 500 nm (enlargement). (G) Immunolabeling for P-Tpr (red) in combination with doublecortin (DCX) (green, marker for neuroblasts) in the subgranular zone (SGZ) of the hippocampus in adult wild-type (WT) mice. Enlargements indicate a DCX+ cell with nuclear P-Tpr and a DCX−cell with no nuclear P-Tpr. Quantification of nuclear or nuclear envelope P-Tpr localizations in Ki-67+, DCX+, and NeuN+ cells in the SGZ of the hippocampus in adult WT mice (n = 4 mice, 79–98 single cells were analyzed per Ki-67, DCX, or NeuN condition). Scale bars: 3 µm, 5 µm (enlargement). Values are the mean ± SEM (p-values calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, * p < 0.05, ** p < 0.01) (H) Electron microscopy determining the localization of Tpr and P-Tpr in comparison to FG-Nups in NSPCs of the hippocampal SGZ in adult WT mice. Quantification of nuclear or nuclear envelope Tpr- and P-Tpr-IR in NSPCs of the hippocampal SGZ in adult WT mice (n = 3 mice, 27–30 single cells were analyzed per Tpr or P-Tpr localization). Scale bar: 100 nm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, * p < 0.05, ** p < 0.01).