Extended Data Fig. 3. Related to Figure 4.

(a) Deletion of HSF-1 abolishes expression of heat shock responsive genes. High density HEK293A WT and HSF-1 KO cells were subjected to heat shock for 1 or 2 h, and mRNA expression of downstream target genes was measured by RT-PCR. Data are presented as mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test, ns, not significant. Two independent KO clones are shown.

(b) Chemical chaperones, which can prevent protein misfolding, cannot block YAP dephosphorylation by heat shock. HEK2933A cells were pretreated with indicated chemical chaperones sodium 4-phenylbutyrate (4-PBA) or DMSO for 24 h and were then subjected to heat shock as indicated. The phosphorylation of YAP was detected by a phos-tag gel.

(c) Knockout of HSP90, but not HSF-1 or HSP70, delays YAP dephosphorylation in response to heat shock. HEK293A WT, HSF-1 KO, HSP70-1/2 DKO, and HSP90α/β DKO cells were subjected to heat shock. Samples were analyzed by western blot on phos-tag gel for YAP phosphorylation and regular gel for YAP protein levels.

(d) Quantification of the Western blots in Extended Data Figure 3c.

(e) The verification of HSP90α and HSP90β knockdown efficiency by Western blot. Two independent siRNAs were used. Immunoblotting in panels b, c and e has been performed two times with similar results. Source data are available online.