Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells
Figure 3
Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.
3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.