Hsf1 regulates cell size in response to stress.
(A) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells at 37 °C as measured by luciferase reporter assay (n = 3 biologically independent samples and 2 experimental replicates each time). (B) Immunoblots of Hsf1 in the cytosolic and nuclear fractions of HEK WT and Hsp90α/β KO cells (α KO, Hsp90αKO; β KO, Hsp90βKO). GAPDH and lamin B1 serve as loading controls (representative blots of n = 2 biologically independent experiments). (C) Fold change of Hsf1 activity of HEK WT, A549 WT, and their respective Hsp90α/β KO cells in chronic HS as measured by luciferase reporter assay (n = 3 biologically independent samples, and 2 experimental replicates each time for HEK; n = 3 biologically independent samples, and 4 experimental replicates each time for A549). (D) Volcano plots of the normalized fold changes in protein levels of some core Hsf1 target genes (list obtained from https://hsf1base.org/) in chronic HS, determined by quantitative label-free proteomic analysis of Hsp90α/β KO and WT HEK cells. Molecular chaperones, whose expression is regulated by Hsf1, are excluded from this dataset. Each genotype was compared with its respective 37 °C control (n = 3 biologically independent samples). Log2 fold changes of > 0.5 or < -0.5 with q-values (adjusted p-values) of < 0.05 were considered significant differences for a particular protein. (E) Flow cytometric quantification of cell size of HEK, A549, and RPE1 cells upon overexpression of WT Hsf1 (with plasmid pcDNA-Flag HSF1 wt) or mutant Hsf1 (with plasmid pcDNA-Flag HSF1 C205; retaining only the first 205 amino acids)(Kijima et al., 2018), and with plasmid pcDNA3.1(+) as empty vector control. Transfected cells to be measured were identified on the basis of their coexpression of EGFP (n = 4 biologically independent experiments). (F) Flow cytometric quantification of cell size in chronic HS after knockdown of Hsf1 in HEK WT, A549 WT and Hsp90αKO cells. Here the chronic HS for A549 cells is at 39 °C instead of 40 °C to reduce HS-induced damage in Hsf1 knockdown conditions (n = 3 biologically independent samples). For all bar graphs, the statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.