The regulatory domain of HSF1 is involved in the pro-apoptotic response to TNF. (A) Upper panel, functional domains and potential DAPK phosphorylation.

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The regulatory domain of HSF1 is involved in the pro-apoptotic response to TNF. (A) Upper panel, functional domains and potential DAPK phosphorylation motifs in human HSF1. The regulatory domain of HSF1 is involved in the pro-apoptotic response to TNF. (A) Upper panel, functional domains and potential DAPK phosphorylation motifs in human HSF1. DBD, DNA-binding domain; HR-A/B, harbour domain/leucine zipper trimerization domain; RD, regulatory domain; C, heptad repeat region C; AD, activation domain. Blue arrows show transcription-inhibitory phosphorylation; red arrows show transcription-inducing phosphorylation; white arrows indicate residues for which phosphorylation has unknown effects on gene transcription. Lower panel, schematic representation of the HSF1 cluster deletion mutant (CDM), containing partially excluded sequence of the regulatory domain (amino acids 250–370). WT, wild type. (B) HCT116 cells were transfected with 2 µg of HSF1-CDM–HA construct. Equal amounts of protein were resolved by SDS-PAGE and probed with the HSF1-specific antibody. The asterisk indicates endogenous HSF1. GAPDH was used as a loading control. Ctrl, control. (C) HCT116 cells were co-transfected with 1 µg of HSF1-CDM–HA and DAPK–FLAG constructs and treated with 0.66 ng/ml of TNF for 12 h. The subcellular localization of exogenous pHSF1Ser230 (red) and DAPK (green) was visualized by confocal microscopy. DAPI staining (blue) was used to identify nuclei. Arrows indicate fragmented chromatin. Scale bar: 12 µm. n = 2 for all experiments. (D) Scatter plots of flow cytometric analysis of annexin-V and propidium iodide staining of untreated HCT116 cells (Ctrl), HCT116 cells subjected to TNF treatment (TNF) and HCT116 cells transfected with either HSF1-CDM–HA cDNA (CDM) or empty vector (EV). TNF treatment was performed at the same time for all samples, 6 h after cDNA transfection, for 24 h. The apoptosis rate is determined as the sum of the lower right quadrant (early apoptosis) and upper right quadrant (late apoptosis). (E) Quantification of the mean annexin-V and propidium iodide fluorescence of the cell population (n = 3; ±s.e.m.). Natalya Benderska et al. J Cell Sci 2014;127:5273-5287 © 2014. Published by The Company of Biologists Ltd