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. 2019 Sep 23;20(11):e48150. doi: 10.15252/embr.201948150

Figure 7. XPO1_S1055 phosphorylation is required and sufficient for YAP nuclear exit at high confluency.

Figure 7

  1. XPO1 and STK38 are required for YAP1 nuclear export at high confluence. A549 cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT) at low or high confluency. Forty‐eight hours later, cells were incubated overnight in the presence of XPO1 inhibitors KPT‐185 and KPT‐330 (final concentration = 1 μM) or with DMSO for all other conditions. The next day, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from three independent experiments, mean ± SEM; ***P < 0.001, Mann–Whitney test). XPO1 activity inhibition by both KPT‐185 and KPT‐330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence (see Fig EV5A for representative images and Appendix Fig S7A for STK38 silencing).
  2. XPO1_S1055 phosphorylation is required and sufficient for YAP nuclear exit. Genome‐edited XPO1 HAP1 cells were cultured for 2 days at low versus high confluency. Cells were then fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***P < 0.001, Mann–Whitney test). Here, phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence, whereas phosphomimetic variants (S1055D and S1055E) potentiated YAP1 nuclear exit (see Fig EV5B for representative images).
  3. Genome‐edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non‐targeting siRNA (siNT)) at low or high confluence. Seventy‐two hours later, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n = 150 cells from two independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt XPO1 (see Fig EV5C for representative images and Appendix Fig S7D for STK38 silencing).