Figure 1. Heat shock activates YAP.
(a) Heat shock induces YAP dephosphorylation. 8×105 HEK293A and A549 cells (human lung cancer) per well were seeded onto six well plates. After 24 hrs, the cells were heat shocked at 43°C for the indicated times and cell lysates were collected for immunoblot and YAP phos-tag gel analyses.
(b) YAP dephosphorylation by heat shock is rapidly reversible. High density HEK293A and A549 cells were heat shocked at 43°C for 30 min or 1 h, then were allowed to recovered at 37°C for the indicated times. Samples were collected for YAP phos-tag gel analyses.
(c) Heat shock induces YAP/TAZ nuclear localization. HEK293A and A549 cells were heat shocked at 43°C for 30 min and then fixed and stained for immunofluorescent microscopy with an anti-YAP/TAZ antibody. Representative pictures from three independent samples are shown. Scale bars, 10 μm.
(d) Quantification of YAP/TAZ nuclear and cytosolic localization. Data were mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test.
(e) Heat shock induces YAP target gene expression. High density HEK293A and A549 cells were subjected to heat shock for the indicated times. CTGF and CYR61 mRNAs were measured by RT-PCR. Data are presented as mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test.
(f) Heat shock overrides other Hippo activating signals to induce YAP phosphorylation. HEK293A cells under medium confluence (4×105 cells per well seeded onto six well plates 24 h before the treatment) were treated with various YAP-inhibitory signals and environmental stresses for 1 or 2 h and then subjected to heat shock for the indicated times. YAP dephosphorylation was determined by phos-tag gel analyses. Immunoblotting in panels a, b and f has been performed two times with similar results. Source data are available online.