Fig. 5

Specific up-regulation of a group of genes by vertebrate HSF1 proteins. (A) Human HSF1 KO cells expressing cHSF1 or cHSF3. Cell extracts were prepared from wild-type (WT) cells, HSF1 KO OUMS-36T-3F (KO1) cells, and HSF1 KO cells stably expressing cHSF1 or cHSF3, and aliquots were subjected to Western blotting. Original blots are presented in Supplementary Fig. S5I. (B) Volcano plots of differentially expressed genes. Microarray analysis was performed using total RNA isolated from the cells described in (A). Genes up-regulated and down-regulated by hHSF1 (WT), cHSF1 (KO + cHSF1), or cHSF3 (KO + cHSF3) were identified and indicated as red and green dots, respectively (fold change >  + 1.3 and < − 1.3, P < 0.05; n = 3). (C) Venn diagram showing genes up-regulated by hHSF1, cHSF1, and cHSF3. A total of 130 genes up-regulated by three HSF proteins are indicated in blue, and 62 genes up-regulated by two HSF1 proteins, but not by cHSF3, are indicated in red. (D) Heat map showing 192 genes significantly up-regulated genes by two HSF1 proteins. Representative genes specifically up-regulated by two HSF1 proteins, but not by cHSF3, are indicated by red lines on the right. (E) Classification of 62 genes specifically up-regulated by two HSF1 proteins according to gene ontology categories. Numbers of genes in each category are indicated. (F) Expression levels of representative genes specifically up-regulated by two HSF1 proteins. Means of normalized mRNA signal values (Log 2) in wild-type (WT) cells, HSF1 KO cells, and HSF1 KO cells stably expressing cHSF1 or cHSF3 are shown (n = 3). Each value is indicated as a dot. (G) Validation of G0S2 expression in HSF1 KO cells expressing HSF1 or HSF3 proteins. G0S2 mRNA levels in various cell lines described in Fig. 3 were quantified by RT-qPCR, and their levels relative to that in wild-type (WT) cells are shown. Data are shown as mean ± SD (n = 3). Asterisks indicate *P < 0.05 and **P < 0.01 by a one-way ANOVA, followed by the Tukey–Kramer test. (H–J) Overexpression of G0S2 partially promotes the proliferation of HSF1 KO cells under daily temperature fluctuations. HSF1 KO cells stably overexpressing G0S2 tagged with HA (clone #1 and #2) were generated (H). These cells were grown for 14 days under daily temperature fluctuations (39.5–30 °C). The number of viable cells was counted, and fold changes are shown (I). Doubling time ratios of each cell line to that of wild-type cells are shown (J). Data are shown as mean ± SD (n = 3). Asterisks indicate *P < 0.05, **P < 0.01, and ***P < 0.001 by a two-way ANOVA (I) or by a one-way ANOVA, followed by the Tukey–Kramer test (J). Original blots are presented in Supplementary Fig. S5I.