Supplementary Figure 4: Stress-induced TEAD inhibition uncouples YAP localization and dephosphorylation in YAP-driven cancer cells. | Nature Cell Biology

Supplementary Figure 4: Stress-induced TEAD inhibition uncouples YAP localization and dephosphorylation in YAP-driven cancer cells.

From: Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation

Supplementary Figure 4

(a,b) Stress-induced TEAD and YAP/TAZ cytoplasmic translocation in H2373 mesothelioma cells, which have homozygous deletion of NF2. Immunofluorescence showing NaCl stimulation induces TEAD and subsequent YAP/TAZ cytoplasmic sequestration (a), despite constitutive dephosphorylation of YAP as shown by western blot (b). NC, normal condition. (c) Nuclear localization of TEAD-VP16 in the presence of osmotic stress. MSTO-211H cells stably expressing TEAD1/4-VP16 construct were treated with NaCl and stained for immunofluorescence. (d,e) Stress promotes TEAD and YAP cytoplasmic sequestration in YAP-driven uveal melanoma cells. 92.1 cells were treated with YAP-inhibiting stimuli as in Fig. 1a, b. NaCl treatment elicits TEAD and YAP cytoplasmic translocation shown by immunofluorescence (d), despite constitutive dephosphorylation of YAP shown by western blot (e). (f,g) p38 mediates stress-induced TEAD cytoplasmic translocation in UM cell lines. Immunofluorescence shows treatment with SB203580 blocks NaCl-induced cytoplasmic translocation of TEAD in 92.1 (f) and OCM1 (g). (h) p38 expression inhibits colony formation of GNAQ-mutant 92.1 cells but not BRAF-mutant OCM1 cells. (i) Colony growth assay showing osmotic stress inhibits anchorage independent growth of YAP-5SA transformed MCF10A. (j) Expression of p38 reduces target gene expression induced by hyperactive YAP as measured by qRT-PCR. Target gene expression is rescued by constitutively active TEAD. Data are presented as mean from n = 2 independent experiments. (km) Immunohistochemistry staining of TEAD. Negative control staining for pan-TEAD antibody (left) and normal kidney tissue staining with pan-TEAD (right) (k). Nuclear staining of TEAD detected in mouse spleen and lung tissues (l). Cytoplasmic staining of TEAD is detected in tubule cells of normal kidney while nuclear staining is detected in renal clear cell carcinomas derived from transformed tubule cells (m). Scale bars in a, c,d, and f,g are 20 μm. Scale bars in km are 50 μm. Statistics source data are shown in Supplementary Table 1. Unprocessed scans of blots are shown in Supplementary Fig. 5.

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