Fig. 3

Vertebrate HSF1 proteins confer resistance to daily temperature fluctuations. (A) Generation of human HSF1 KO cells expressing vertebrate HSF1 proteins. Cell extracts were prepared from wild-type (WT) cells, HSF1 KO OUMS-36T-3F (KO1) cells, and HSF1 KO cells stably expressing hHSF1, cHSF1, malHSF1, or plaHSF1, and aliquots were subjected to Western blotting using anti-HSF1 (anti-mHSF1s) or anti-β-actin antibody. Original blots are presented in Supplementary Fig. S5D. (B) Expression of HSPs in HSF1 KO cells expressing vertebrate HSF1 proteins. Cell lines described in (A) were untreated (C) or treated with heat shock at 42 °C for 6 h (H). Extracts of these cells were prepared and subjected to Western blotting. Two isoforms of HSP110 are indicated by arrowheads. Original blots are presented in Supplementary Fig. S5D. (C) Expression of vertebrate HSF1 proteins restored the proliferation rate of HSF1 KO cells. Cell lines described in (A) were grown at 37 °C for 3 days. The number of viable cells excluding trypan blue was counted, and fold changes are shown. Data are shown as mean ± SD (n = 3). Asterisks indicate *P < 0.05 by a one-way ANOVA, followed by the Tukey–Kramer test. (D,E) Proliferation of the cell lines under daily temperature fluctuations. Cell lines described in (A) were grown for 14 days under daily temperature fluctuations between cold (30 °C) and warm (39.5 °C) temperatures. The number of viable cells was counted, and fold changes are shown (D). The ratios of doubling time of each cell line to that of wild-type cells are also shown (E). Data are shown as mean ± SD (n = 3). Asterisks indicate ***P < 0.001 by a two-way ANOVA (D) or by a one-way ANOVA, followed by the Tukey–Kramer test (E). (F) Colony formation of the cell lines exposed to daily temperature fluctuations. The cells, which were grown for 14 days under the conditions as described in (D), were re-plated in 60-mm culture dishes for 10 days. The number of colonies were counted, and the percentages of colonies originating from single cells (plating efficiency) is shown. Data are shown as mean ± SD (n = 3). Asterisks indicate ***P < 0.001 by one-way ANOVA, followed by the Tukey–Kramer test.